BIOCHEMISTRY - CORE

POLYCLONAL ANTIBODY-MEDIATED STABILIZATION

t_ OF SOME USEFUL ENZYMES (IMMOBILIZATION OF RESTRICTION ENDONUCLEASES)

THESIS SUBMITTED FOR THE DEGREE OF

doctor of ^tjilosopljp

BIOCHEMISTRY

emiata Varsl^fney

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2000

^ * - ^ : : ) - ; > '0Q2

, ^ . ^ ,

r A

^ Acc, No )^jl

/y. >

I 3 MAR 2002

M A • iCCC

> : ) r

v^5^?n

T5376

^Dedicated 'To niij T'arenu

Who gave nie Wisdom and

jinurag for making it possible

Certificate

/ certify that the work presented in this thesis entitled "'Polyclonal

antibody -mediated stabilization of some useful enzymes" has

been carried out by Jiemlata Varshney under my supervision. It is

original in nature and has not been submitted for any other

degree.

Vi^r M. MeemuddinP ^'^ 'Professor

CONTENTS Page No.

List of Abbreviation (i) List of Illustrations (ii) ListofTables (v)

1. Introduction LI Restriction Enzymes 1

L L l Nomenclature 3 LL2 Classification 3 LL3 Type II Restriction Endonucleases 7

1.L3.1 Physico Chemical Properties 9 LL3.2£coRI 10 l.l3.3BamHl 13

L2 JM109 14 1.3 Enzyme Immobilization 16

1.3.1 Use of monoclonal/polyclonal antibodies 19 1.3.2 Enzyme immobilization with the help of antibodies 23

1.3.2.1 Oriented immobilization of antibodies 25 1.3.2.1.1 Immobilization on metal affinity support 26

1.3.3 Immobilization of restriction enzymes 29

2. Experimental 2.1 Material 34

2.1.1 Chemicals 34

2.1.2 Bacterial Strains 35

2.1.3 Media 36 2.2 Methods 36

2.2.1 Cell cultures and preparation of lysates 36 2.2.1.1 Cultivation of f.co/i pMB4 36

2.2.1.1.1 Cell lysis 37 2.2.1.2 Cultivation of E.coli JM109 37

2.2.1.2.1 Preparation and fractionation of crude bacterial lysate 38 2.2.2 Immunological procedures 39

2.2.2.1 Preparation of antigens 39 2.2.2.2 Immunization 40 2.2.2.3 Oucterlony double diffusion 40 2.2.2.4 Isolation and purification ofIgG from the antisera 41

2.2.2.4.1 Ammonium Sulphate fractionation 41 2.2.2.4.2 DEAE cellulose chromatography 41

2.2.3 Immobilization of Antigen/Antibody 42 2.2.3.1 Immobilization of IgG on Sepharose 4B 42 2.2.3.2 Immobilization of human serum albumin on CNBr activated

sepharose 4B 43 2.2.3.3 Oriented Immobilization of Antibodies 43

2.2.3.3.1 Immobilization of anti5amHI IgG on metal affinity support 43 2.2.3.3.2 Immobilization of antiHSA IgG 44

2.2.4 Enzyme Immobilization 45 2.2.4.1 Immobilization ofEcoRl/BamHl on IgG Sepharose 45 2.2.4.2 Immobilization of 5amHI on IgG Co^^-IDA Sepharose 45 2.2.4.3 Immobilization of ^coRI from bacterial lysate 46 2.2.4.4 Affinity Immobilization of SpAii^coRI 46

2.2.4.4.1 Immobilization on goat IgG- Sepharose 4B 46 2.2.4.4.2 Immobilization on favourably oriented IgG 47

2.2.5 Estimation of Proteins 48 2.2.6 Electrophoresis 48

2.2.6.1 Non-denaturing polyacrylamide gel electrophoresis 48 2.2.6.2 SDS PAGE 49 2.2.6.3 Staining procedure 50 2.2.6.4 Agarose gel electrophoresis 50 2.2.6.5 Staining of DNA in agarose gel and visualization 51

2.2.7 Assay of Restriction endonucleases 51

\. Immunoaffinity Immobilization ofEcoRl 3.1 Immunization of rabbits against^coRI 52 3.2 Immunoaffinity Immobilization of £coRI 52

3.2.1 Behaviour of immobilized EcoRI 56 3.2.1.1 Restriction activity on plasmids 56 3.2.1.2Stability of the immobilized £coRI 56

3.2.1.2.1 Effect of exposure to various temperatures 56 3.2.1.2.2 Storage stability and reusability 59

3.2.2 Direct Immobilization of fcoRI from the curde lysate of£'.co/jpMB4 59

L Immobilization of BamHl on antienzyme antibody supports 4.1 Immunization of rabbits and isolation of IgG from the antisera 64

4.2 Immobilization of BamUl on Sepharose 4B matrix precoupled with

anti^flmHIIgG 68 4.2.1 Behaviour of the immunoaffinity immobilized 5fl/wHI 71

4.2.1.1 Action on plasmids 71 4.2.1.2 Effect of temperatures 71 4.2.1.3 Storage stability and reusability... 74

4.3 Properties of amHI immobilized on Sepharose matrices bearing randomly coupled or favourably oriented antibodies 74 4.3.1 Action on plasmids 78 4.3.2 Effect of temperatures , 82

5. Affinity immobilization of genetically engineered hybrid protein 5.1 The Recombinant organism E.coli JM109 expressing the fusion

protein SpAirfcoRI 84 5.2 Preparation of immobilized goat IgG 84 5.3 Fractionation of £.co/iJM109(3P)lysate 84 5.4 Affinity immobilization of SpA:i^coRI 86

5.4.1 Activity on plasmids 90 5.4.2 Stability of the immobilized preparation 90

5.5 Oriented immobilization of the fusion protein 90

6. Discussion 96

7. Summary 106

8. Bibliography 108

List of Abbreviations

BSA CONA DEAE IDA IgG PAGE SDS LB NB E. coli M9 Asp Glu Lys

Bovine Serum Albumin ConcanavalinA Diethyl ethylaminoethyl cellulose Iminodiaceticacid Immunoglobulin Polyacrylamide gel electrophoresis Sodium dodesyl sulphate Luria Bertani Nutrient Broth Eschreschia coli Minimal media Aspartarate Glutamate Lysine

List of Illustrations

Page No.

1.1 The EcoRl and BamHl recognition site 11

1.2 Principal modes of enzyme immobilization 20

1.3 Principle of enzyme immobilization by metal chelate binding with regeneration of the carrier 27

3.1 Immunodiffusion of anti EcoRl antiserum against 53 EcoRl

3.2 Immobilization of EcoRl on the immunoaffinity support 55

3.3 Digestion of the plasmid pBR322 and pBHLUCP by soluble and antibody support immobilized EcoRl 57

3.4 Effect of pretreatment at various temperatures on soluble (A) and antibody support immobilized (B) preparations of EcoRl 58

3.5 Effect of storage on the restriction activity of EcoRl immobilized on the antibody Sepharose 60

3.6 Reusability of the EcoRl immobilized on antibody support 61

3.7 Time dependent cleavage of DNA by E. coli pMB4 lysates

(A) and the immobilized preparation (B) 63

4.1 Immunodiffusion of rabbit antiserum against BamHl 65

4.2 Isolation of gamma globulin from sera of rabbit immunized with the restrictionenzyme BamHl 66

4.3 Molecular weight determination of IgG 67

4.4 Immobilization of BamHl on the anti BamHl IgG Sephrose support 70

4.5 Digestion of the plasmids pBR322 and pUC19 by soluble and antibody support immobilized BamHl 72

4.6 Effect of pretreatment at various temperatures of soluble (A) and antibody support immobilized (B) preparations of BamUl 73

4.7 Storage stability of immobilized BamHl at 4°C 75

4.8 Reusability of the BamHl immobilized on antibody support 76

4.9 Immobilization of BamHl on supports bearing comparable amounts of anti BamHl IgG linked to cyanogen bromide activated Sepharose (A) or bound on cobalt charged IDA Sepharose (B) 79

4.10 Concentration dependent cleavage of X, DNAby immobilized BamHl preparation 80

4.11 Digestion of the plasmids by BamHl linked to IgG immobilized on CNBr activated (A) and metal chelated support (B) 81

4.12 Effect of pretreatment at various temperatures of BamHl immobilized on IgG-Sepharose (A) or IgG CO^^-IDA Sepharose (B) 83

5.1 EcoRl activity in the lysates ofE. coli JM109 87

5.2 Ammonium sulphate fractionation of the lysate oiE. coli

JM109 88

5.3 Immobilization of fusion protein fcoRIirSpA on randomly

coupled IgG support 89

111

5.4 Digestion of plasmids pBR322 and pUC19 by soluble 91 and antibody support immobilized fusion protein

5.5 Effect of pre treatment at various temperatures on activity of immobilized EcoRl 92

5.6 Immobilization of EcoRl::SipA on anti HSA IgG favourably oriented anti HSA IgG on HSA Sepharose 94

5.7 Cleavage of plasmids with soluble and EcoRhiSpA immobilized on favourably oriented IgG support 95

List of Tables

Page No.

Table 1.1 Some characteristics of Restriction Endonucleases

Table 1.2 Plasmids present in the E. coli JM109

Table 1.3 Enzymes immobilized favourably with the help of antibodies

Table 3.1 Binding of partially purified IgG fraction on CNBr activated Sepharose

Table 4.1

Table 4.2

Table 5.1

Binding of purified IgG fraction on CNBr activated Sepharose

,2+ Binding of IgG on Co -IDA Sepharose

Binding of purified IgG on HSA Sepharose

5

15

22

54

69

77

85

ACKNOWLEDGMENTS

I take this privilege to express my most sincere and profound gratitude to my supervisor, Prof. M. Saleemuddin for his excellent guidance, forbearance, sustained interest and trust through out the course of this study. His few but eloquent words never failed to guide me to the right path.

I wish to record my grateful thanks to Prof. S. M. Hadi Chairman Department of Biochemistry for providing facilities for research work. Kind help of my teachers. Dr. J . Iqbal, Dr. J.Mussarat, Dr. R. Mehmood, Dr. A. N. K. Yusufi, Prof. M. Ahmad, Dr (Ms) N. Bono, (Ms) B. Bono and Dr. Q. Hussain within their busy and varied schedule \s gratefully acknowledged.

I am highly thankful to Prof. K. Schugerl and J . I . Rhee of University of Hannover, Dr. Arshad Rehman of Chicago University and Prof. H.K. Dass of J.N.U for providing valuable strains and plasmids for the study.

I owe a word of deep appreciation to all my seniors with special mention of Dr. Mana, Dr. Fahim, Dr. Sandeep and Dr. Shakeel for their cooperation and encouragement.

My heartfelt thanks are extended to my friends Atia, Maria, Shaukat, and Waseem, for moral support and valuable help extended by them as and when needed.

Cheerful ass\s\ar\ce, from Pawan, Sonish, Arshi, Hina and Mansour, is thankfully appreciated. Thanks are due to my lab colleagues Minhaj, Jahangir, Saurabh, Farhan, AmeS, Am/r, \J\fa\, Sabiha, Samma, Sadaf, Athar, Moyad, Sonali, Rana, Tariq, Kashif and Shahid.

I also wish to thank Asad, Salman, Mushahid, Hina, Yogesh and Matin of Interdisciplinary Unit of Biotechnology A.M.U. and Shaba of Insti tute of Agriculture A.M.U for their selfless help.

Words are insufficient to express my infinite gratitude to my parents for their unending support and constant inspiration through out my carrier. I also deem it necessary to acknowledge my parents in law, my brother Nirvesh and sisters Binni and Gunjan whose affection and support have always been stimulating at the time of my need.

Finally, all my efforts are animated because of Anurag. Without his patience and favorable anchor my present endeavor would not have succeeded.

Appreciations are also due to Mujtaba Sahib and to non-teaching staff of the department especially Khan Sahib for their help.

Lastly Council of scientific and Industrial Research is gratefully

acknowledged for the providing financial assistance.

[Hemlata Varshney]

UntroducHon

R; 1.1 Restriction Enzymes

estriction enzymes, also known as restriction endonucleases

(REs) function in the host-controlled restriction

modification system occurring principally in bacteria

(Anderson, 1993). They are strain specific enzymes that can act as

primitive immune systems and rapidly destroy foreign DNAs by

catalyzing the endonucleatic cleavage of sequences that lack the species-

specific double stranded fragments with terminal 5'phosphates. The REs

thus achieve the destruction of any foreign DNA that invades the host

cell (Modrich, 1982; Modrich and Robert, 1982; Bennet and Halford,

1989).

In addition to the restriction enzymes, each of the bacterial strains

possesses specific DNA methylases that transfer methyl groups from S-

adenosylmethionine (AdoMet) to specific adenine or cytosine residues in

the DNA. In this way, the DNA of the cell is protected against its own

restriction enzymes.

Although bacteria remains the prime source of REs - over 2400

REs with more than 200 specificities have been reported to date, few

reports of their occurrence in eukaryotes are also available. Few lower

eukaryotes like Saccharomyces cerevisiae (Watabe et al., 1981), Pichia

membranefaciens (Watabe et al., 1984) and Chlamydomonas reinhardtii

(Burton et al, 1977) contain DNA site-specific endonucleases but they

differ from bacterial REs in lacking the unique recognition sequence. In

some instances, the restriction modification system can be induced in the

eukaryotic host upon phage infection (Xia et al, 1986a; Xia et al.,

1986b). Spirulina platensis, a multicellular filamentous, spiral blue green

alga has been reported to produce restriction enzymes (Kuwamura et al.,

1986). An interesting report on Hsal, a restriction endonuclease of

human origin is also available (Lao and Chen, 1986).

Sequence specific protein DNA interactions play an essential role

in the recognition by a protein of its cognate binding site on DNA. The

restriction modification system is an attractive model system in the study

of such interactions in view of the simple and sequence specific nature of

the reactions catalyzed. One of the most far-reaching applications of

restriction endonucleases is the in vitro construction of DNA molecules

with novel biological activities. With the help of appropriate REs,

segments of DNA can be excised, added or rearranged in a given

genome (Lai and Nathans, 1974). Alternatively, DNA from diverse

sources can be joined to yield artificial recombinant molecules with the

help of REs (Jackson et al, 1972; Lobban, 1973; Hershfield, 1974;

Sambrook et al, 1989). Such restructured molecules can then be cloned

in suitable cells (Kerft and Hughes, 1982) and propagated to yield large

quantities of the new DNA and in some cases even the specific gene

products (Hershfield et al., 1974; Murray and Murray, 1974). An

understanding of the reaction mechanism of these interacting enzymes is

not only important in the area of restriction and modification but is also

increasingly relevant to other important biological reactions; such as

genetic recombination, DNA splicing and transportation of genetic

elements. Because of the nature of their controllable, predictable,

infrequent and site-specific cleavage of DNA, restriction endonucleases

have proved to be extremely useful as tools in dissecting, analyzing and

reconfiguring of genetic information at the molecular level (Rosenberg,

1991).

1.1.1 Nomenclature

The discovery of large number of restriction/modification enzyme

systems necessitated a universally acceptable nomenclature and one such

was proposed initially by Smith and Nathans in 1973. The specific name

of the host organism is identified by the first letter of the genus name and

the first two letters of the species to form a three-letter abbreviation in

italics. For example, Escherichia coli is abbreviated as Eco. The strain or

type identification follows without italics, for example EcoRl where R is

the strain type. When a particular host strain has more than one

restriction and modification system, Roman number, for example, Bgl\,

and 5^/11, identifies them.

1.1.2 Classification

The restriction enzymes were originally classified into two types,

based on the complexity of their structure and cofactor requirements

(Boyar, 1971). As information on the structure and behaviour of REs

accumulated, additional criteria were employed to differentiate between

various restriction and modification systems and a third class of

restriction enzymes was identified. Restriction/modification enzymes

system is presently divided into three categories- Type I, Type 11 and

Type III (Smith, 1979; Yuan, 1981). While Type I and Type III enzymes

carry both restriction and methylation properties in one protein, usually

comprising of two or three heterologous subunits, Type II enzymes

exhibit only restriction activity. Type I and Type III restriction/

modification enzyme systems occur less commonly and yet they are

biologically interesting. The properties of these three types of restriction

enzymes are listed in Table 1.1. Only Type I enzymes form complexes

with unmodified DNA, which are large enough to be retained on

nitrocellulose filters (Horiuchi et al., 1975). The Type I REs, such as

those from E. coli B and E. coli K, and the Type III enzymes, such as

those coded by the phage PI and the plasmid P15, are multifunctional

proteins capable of both cleavage and methylation of the unmodified

DNA. They comprise of three (Type I) or two different subunits (Type

III) and require ATP and Mg^* for the catalytic activity. Although

AdoMet is essential for restriction by Type I enzymes, it only stimulates

the action of those of Type III.

Type II enzymes (Wells et al., 1981), best represented by EcoRl

(Heitman, 1992), appear to have far simpler protein structure (e.g. EcoKl

is a homodimer) and require only Mg ^ for activity. DNA modification is

catalysed by a separate methylase in E. coli that is active under the

restriction conditions.

The susceptibility of a particular DNA to a restriction and

modification system depends on the presence of certain nucleotide

sequences (host specific sites). In general, the sequences recognised by

Type II enzymes consist of 4 to 6 bases and have two-fold symmetry

(Newman et al, 1994). Several restriction enzymes are able to recognize

1 ^

a 1

NN

^

1

_____

0

o o c

 N C

o >" c ^ a >-.

cd "^ OH T 3

C/D

ed

O

3 • 1 - H

6

C/3

0) • l - H -4—4

>

<

o • 1—( +-> cd

• t - H

-a o

e cd C o

• i - H

C/3

OS

T — (

cd

(U

N

c

Hi

-a o ^

00

1/3

c ;3

3

•4-J

c

T3

H

kH

2 00 c

o l - l (X

(N

o -a

+

CU

5

+

S

CLH"

<

o •a <

o

!/3 <1>

- 4 - ^

c 

• 4 — "

o • ^ H

y

OH 00

•1—>

ai O

a: o 8

a-O)

vn

-4-J

(/3 O

^4-1

o CO

O

X) VO (N

1

-4—*

1/3 O

-4-J

Cd

I d kH

a 4)

O

"cd

6 o cd >-(

X • ^ H 1/3 1/3

u c2

!/3

• — 4

C/5 0)

cd > cd 

• r H 1/3

-4—> • ^ H

o O CD OH

1/3 O

J3

H O

X o o o T — 1

-4—> t/3 Cd

+

D cd CI,

O ^ !/3 D O T3 .C O >H Z < C/5 K

!/3 0) >

 o B 13

H o I d

s N C3

PJ

r

.2 >

^ o D -Ji OH

o S t O T3 g " O Z < C/3 PC

+ ^ba o B S. . '^ CLH ^ . t i

i i ^ &1 5 cd on

' .2 "5 " ai ^ X O JH < S K

.2 c

>-. "cd

•J^ kH 5 '-C

y = -i 1/) -T^

C « C 1)

2 6 .2 1

Q 0 oi oo

00 cK 2 ;^

00 ON

C cd

;^

o i-( =3 O

C O

a specific sequence but cleave at a site 5 to 9 bases away. In case of

other REs, the sequence being recognized has a hyphenated structure.

Both endonucleolytic cleavage and methylation take place within these

sequences. In contrast, the Type I and Type III sites are asymmetric. In

Type III, the sequence consists of 5 to 6 bases, while in Type 1 the

sequence are hyphenated with two constant domains of 3 and 4 bases

separated by a non specific spacer of 6 or 8 bases. Although recognition

and methylation take place at these sites, DNA cleavage occurs

elsewhere 24 to 27 bases away in Type III or up to several thousand

bases away in Type I REs (Yuan, 1981).

ATP and Mg^* stimulate methylation in the Type I and Type III

but not in Type II REs, whereas in Type I systems, the enzyme can

catalyse either restriction or modification, but the two activities are

mutually exclusive. Type III enzymes on the other hand are able to

express both activities simultaneously. The modulation of enzymatic

activity can be caused by the relative concentration of ATP and AdoMet

(Type III) or by the interaction of the protein with the host specific DNA

sites (Type I). In the later cases, the site acts as an allosteric effector,

enabling the enzyme to cut the DNA a short distance away (Type III).

Alternatively, ATP hydrolysis is coupled to a novel mechanism of DNA

translocation that brings the enzyme into contact with potential cleaving

sites far removed from the recognition site (Type I) (Modrich, 1979).

New developments in this area have resulted from studies on the gene

structure of the restriction and modification loci and their transcription

patterns, the nucleotide sequence of the host specificity sites, and the

multiple steps involved in the interaction between the enzymes and the

DNA (for review see Lesser et al., 1993).

1.1.3 Type II Restriction Endonuclease

Protein-DNA selectivity is the central event in many biological

processes and Type II restriction endonucleases are attractive systems

for the study because of their high specificity and remarkable variety

(Roberts and Halford, 1993; Pingoud and Jeltsch, 1997). Despite the

diversity of sources and specificity, more than 2400 Type II REs

representing 188 different specificities have now been discovered and

studied (Roberts and Macelis, 1993). These enzymes are homodimers

with no primary sequence homology, despite appreciable homology in

the protein folds (Aggarwal, 1995).

The Type II REs generally recognize specific oligonucleotide

sequences, which vary between 6 to 8 bases and make double stranded

cleavage, that generate the unique DNA fragments. They are capable of

non-specific binding to DNA and make use of linear diffusion to reach

their target site. Binding and recognition of the specific sites involve

contacts with the bases of the recognition sequence and the

phosphodiester backbone over approximately 10 to 12 base pairs.

Specific binding is accompanied by conformational changes both in the

protein and the DNA. This mutual induced fit leads to the activation of

the catalytic centres (Lesser et al., 1993).

The precise mechanism of cleavage has not yet been established

for any restriction endonuclease although two have however been

proposed- the substrate assisted mechanism and the two metal ion

mechanism (Grabowaski et al., 1996). Only the former appears to be

operative in case of EcoRl action and as there are only two acidic

residues (Asp-91 and Glu-111) in the catalytic centre that can bind only

one Mg^* ion, the two metal ion mechanism can be ruled out. In BamHl

two divalent metal ion binding sites have been clearly assigned to the

catalytic centre along with substrate assisted mechanism. Structural

similarities between EcoRl, EcoRV, BamHl, Pvull suggest that several

Type II REs may be not only functionally but also evolutionarily related

(Pingoud and Jeltsch, 1997).

The RE recognition sites are centred around a core DNA, di or

tetra nucleotides that permit identification of both isoschizomers and

specific sequence of interest. Their sequence specificity is remarkable.

For example the activity can be million times lower as the result of a

single base pair change within the recognition sequence (Roberts and

Halford, 1993). This remarkable specificity is crucial for the prevention

of accidental cleavage at non-specific sites in a DNA sequence.

The use of the Type II enzymes has resulted in revolutionary

advances in biological research. In particular the construction of

recombinant genomes has attracted considerable publicity and

widespread debate. This system also provides an excellent subject for the

study of protein-DNA interaction. The detailed study of Type II system

has been focused primarily on the EcoRl and BamHl and excellent

reviews have appeared on the subject (Modrich, 1979; Rosenberg, 1991;

Hietman, 1992).

1.1.3.1 Physico-Chemical Properties

Many REs have not been characterized in great detail for their

physico-chemical properties- apparently more attention has been

diverted towards their applications rather than enzymology. The

structure and mechanism of action of only certain restriction enzymes

has however been examined in detail (Modrich, 1979; Yuan, 1981;

Frederick et al., 1984; Ehbrecht et ai, 1985). Enzyme kinetics and

properties like pi, K„, molar extinction coefficient, and amino acid

composition are available for a few REs only (Green et al, 1975). pH

optima of most of the restriction enzymes lie slightly towards alkaline

side (around 7.5) with a maximum value of 9.5 (Clarke and Hartley,

1979; Bickle et al, 1980) and a minimum value of 7.0 (Polisky et al,

1975; Bron and Horz, 1980). Several REs have been found to be stable

over a wide range of pH (Bron and Horz, 1980). Restriction enzymes of

mesophilic origin possess temperature optima around 37°C while those

from the thermophilies have those ranging between 50-67°C (Bingham et

al, 1978; Lacks, 1980). Interestingly, HaeWl from a non-thermophilic

bacterium is fully active at 70°C, whereas its companion enzyme HaeW

is readily inactivated above 42°C (Blakesley et al, 1977).

All Type II restriction enzymes require Mg^* for activity,

although their optimum concentration requirement varies. Sometimes

Mn " partially substitutes for Mg^* but higher concentrations of the ion

are usually inhibitory (Hsu and Berg, 1978; Tikchonenko et al, 1978).

1.1.3.2 Ecom

Of the many known restriction-modification systems, the

best characterized is £'coRI (EC 3.1.23.13) (Yoshimori et al., 1972).

EcoRl is a symmetric homodimer (Modrich, 1982; Modrich and Roberts,

1982; Bennet and Halford, 1989) that cleave the sequence -GAATTC-

(Fig. 1.1) between G and A on both the DNA strands (Hedgpeth et al.,

1972) generating 3' hydroxyl and 5' phosphoryl termini (Mertz and

Davis, 1972). The EcoRl methylase is a monomer which, despite

lack of homology with the endonuclease, also recognizes the

sequence -GAATTC- and methylates the N^ amino groups of the two

central adenines and prevents cleavage by the endonuclease.

In vitro studies revealed that ^coRI endonuclease initially binds

DNA non-specifically and then scans the molecule in one-dimension to

locate the recognition site, GAATTC (Jack et al., 1982; Terry et al.,

1985), possibly by recognising distorted B form DNA (Dickerson and

Drew, 1981; Dickmann and McLaughlin, 1988; Nerdal et al., 1989;

Lane et al, 1991). The catalytic requirements of the EcoRl are relatively

simple. In vitro restriction requires only unmodified DNA and a divalent

cation while unmodified DNA and AdoMet are sufficient for in vitro

modification (Greene et al., 1974).

Under standard condition, recognition and catalysis occur with

high fidelity. Mg^* is essential for EcoRl activity (Modrich, 1982;

Bennet and Halford, 1989) and may play a role in substrate recognition

by inducing conformational changes required for catalysis (Taylor

10

Fig. 1.1 The EcoRl and BamHl recognition sites.

The endonuclease and methylase recognize the respective

palindromic DNA sequences. Positions of DNA scission and

methylation by EcoRl (A) and BamHl (B) are indicated by

scissors and astericks, respectively.

11

G^AATTC

C T T A A G (A)

G G A T C C

CCTAG.G * oV

(B)

et al, 1990). Recently, EcoKl has been subjected to a battery of

structural,

biochemical, and genetic investigations which revealed that the enzyme

is enclosed with redundant sequence discrimination mechanism that

cooperate in substrate recognition (for review see Rosenberg, 1991;

Heitman, 1992). In addition, the X-ray crystal structure of an £coRl

endonuclease-DNA complex is available at 2.8°A resolution (Kim et al,

1990). The catalytic centre oiEcoKl has been shown to comprise of one

basic (Lys-113) and only two acidic residues (Asp-91 and Glu-111).

These EcoKl enzymes are the simplest sequence specific DNA

enzymes known and are well suited for the study of DNA sequence

recognition by proteins (Heitman, 1992). Furthermore, since the

endonuclease and methylase activities are separate under in vitro studies

(Greene et al, 1978), and do not share a common subunit (Yoshimori,

1972), the system may offer a unique opportunity to compare two

unrelated proteins, which interact with the same DNA sequence.

Several efficient procedures for the isolation, purification of

£coRI are available (Greene et al, 1974; Modrich and Zabel, 1976).

Studies on purified EcoRl show that denatured and reduced

enzyme behaves as a single 28,500 kDa peptide on electrophoresis in

SDS-PAGE. In the native state, however the enzyme exists as an

equilibrium mixture of dimers and tetramers of the 28,500 subunit. The

value of Keq (4xlO'^M) together with results of kinetic analysis indicates

that the dimer is catalytically active. Although it has been suggested that

the tetramers constitutes a significant fraction of the endonuclease within

12

the cell, it is not still clear whether the enzyme in aggregated state

possesses endonuclease activity (Modrich and Zabeel, 1976)

1.1.3.3 BamWL

Another of the Type II site specific deoxyribonucleases BamHl

(EC. 3.1.21.4) also proved to be a useful enzyme because it produced a

single stranded end after cleavage between the guanine bases at the sites

of 5'-GGATCC-3' (Roberts et aL, 1977) as shown in Fig. 1.1 (B),

providing a useful substrate for ligation. In addition a number of existing

or developed cloning vectors like SV40 (Goff and Berg, 1977), pBR322,

pMB9 (Boliver et al., 1977a; Boliver et aL, 1977b) contained a single

recognition site for BamWl. These advantages contributed to the

extensive use of BamRl, in genetic and biochemical studies (Hinsch and

Kula, 1980). Infact BamRl is one of the first discovered and

characterized REs (reviewed by Nardone and Chirikjian, 1987).

Smith and Chirikjian in 1979 examined the structure of BamRl in

detail and reported that the enzyme exists as catalytically active dimers

or tetramers of 22,000 (± 500) kDa subunit. The enzyme has rather a

broad pH -activity profile in Tris-HCl with optimum activity at pH 8.5.

BamHl requires Mg ^ which can be replaced with Mn * with some

decrease in activity but without any alteration in specificity.

The gene coding for Bam\{\- BanMR has been cloned, sequenced

and expressed in E. coli (Brook ef A/., 1989, Brook era/., 1991; Jack

et al, 1991). The recombinant BarnRl protein (24,570 Da) has been

purified to homogeneity, crystallized and its crystal structure determined

(Newman et al, 1994). Genetic and biochemical studies showed that

13

residues Asp-94, Glu-111 and Glu-113 of BamHl are important for the

catalytic function (Xu and Schildkrant, 1991; Domer and Schildkrant,

1994).

BamHl shows striking resemblance in secondary structure to the

endonuclease EcoRl (Kim et ah, 1990; Rosenberg, 1991), despite the

lack of sequence similarity between them. The active site of BamWl is

structurally similar to that of £coRI but the mechanism by which BamHl

activates the water molecule for nucleophilic attack appears to be

different. Overall, BamHl is a far more compact enzyme than ^coRI and

lacks many of the secondary structural elements that interrupt the

common core motif in EcoRl (Aggarwal, 1995).

1.2 JM109 Plasmid harbouring E. coli JM109 strain used in the study carried

a three-plasmid system (pRK248CI, pMTC48, pEcoR4) (Rhee et al,

1994) as listed in Table 1.2. The production of plasmid pMTC48 with

20-100 copies represent expression vector for the fusion protein of

Staphylococcus aureus protein A (SpA) and the restriction endonuclease

EcoRl (SpA::EcoRl). To reduce the toxicity of the fusion protein (as the

over production of £'coRl become toxic to the cell) the host was

introduced with 20-30 copies of another plasmid pEcoR4, which

expresses constitutively the £coRI-methylase. It also carries the

resistance gene against chloramphenicol.

To avoid the load on the host protein machinery as well as the

reaming toxicity of the product during the growth phase, the expression

14

Table 1.2

Plasmids present in the E. coli JM109

General characters

clts857, TcR

Ecorl-methylase,

CmR

SpaiiEcorl, ApR

Size (kbp)

9.2

6.0

4.9

Copy number

8

25

80

Inducible promoters

PR, PlacUVS

Reference

Bernard et al, 1979 Bottermann and Zabeau, 1985

Maschke et al, 1991

- tetracycline, Cm-chloramphenicol, ap-ampicillin

of SPA::£coRI is transcriptionaly regulated by two independent

promoters - Piacuv5 " promoter inducible by isopropyl thiogalactoside

(IPTG) and A,PR -promoter which requires the cl repressor protein for

suppression. The repressor is encoded on the third plasmid pRK248cI in

the form of temperature sensitive cl protein mutant cl857. This plasmid

is a derivative of pRK248 R2 group (Thomas et al, 1979; Maschke et

al., 1992; Brandes et al, 1993). It was generated by cloning the Bgl\

fragment (2392 bp) of X, phage cI1857 Saml into single BglW site of

pRK248 (Bernard et al, 1979). The expression of the fusion protein was

induced by a temperature shift using P^ promoter of X, phage regulated

by the repressor plasmid pRK248cI. Using inducible promoters on the

production plasmids, it is possible to switch on the expression at any

time, resulting in separation of the growth and production stages. The

cells grow in the repressed state with no expression of the cloned gene

product in the first growth stage and thereby minimize the expression

related instability problem. Maintaining selection pressure of tetracycline

in the medium prevents plasmid instability (Maschke et al., 1992).

The plasmid harbouring E. coli JM109 strain grows well on M9

medium as opposed to the plasmid free strains. In LB medium both the

strains propagate well.

1.3 Enzyme Immobilization

In recent years there has been a considerable increase in the use of

enzymes as industrial catalysts in view of the advantages offered by

them over conventional chemical catalyst. Enzyme exhibit high catalytic

16

activity and remarkable degree of substrate specificity and catalyzes

various reactions under mild conditions. Biotechnological applications of

enzymes are limited by the high cost of their production and

stabilization. Most enzymes are readily inactivated by heat, chemicals,

proteases and various other environmental factors. In addition, most

enzymes are soluble in water and their recovery from the reaction

mixture for reuse is problematic and uneconomical. Further more, the

practical applications of enzymes may often necessitate their use under

non-physiological conditions such as elevated temperatures (to increase

the productivity and prevent microbial contamination), in non-aqueous

organic environment (to shift the reaction equilibrium towards desired

product) and at non-optimal pH (for maximum stability).

To overcome such limitations, remarkable attention has been

directed towards the developments of strategies for the improvement of

enzyme stability. These include chemical modification and/or

immobilization of the enzymes, use of additives and searching for the

enzyme of desired stability in extremophiles (Brock, 1985; Saha et ai,

1988; Jafri et ai, 1995; Jafri and Saleemuddin, 1997).

Immobilization on variety of solid supports has also turned out to

be an excellent method of enzymes stabilization and with the remarkable

developments in immobilized enzyme technology, more and more

chemical transformations are being achieved with the help of

immobilized enzymes. Immobilization affords several advantages such

as reusability, enhanced stability and greater control of the catalytic

process. In fact enzyme immobilization has currently become an

important component of enzyme engineering.

17

By definition, an immobilized enzyme is physically localized in a

certain region of space or converted from a water-soluble to a water

insoluble state (Klibanov, 1983; Martinek and Mazhev, 1985; Chibata et

al, 1986). The biotechnological applications of immobilized biocatalysts

encompass several fields of general interest and in particular clinical and

analytical chemistry, medicine, food and pharmaceutical technology,

organic synthesis and industrial production of chemical compounds.

Several useful reviews devoted to these subjects are available (Pitcher,

1980; Klibanov, 1983; Bowers, 1986; Chibata et al, 1986; Gemeiner

etal, 199 A).

Immobilized enzymes may also simulate the state of enzymatic

proteins within the intracellular microenvironment of living cells and

provide a good model system to study and solve some basic problems in

enzymology (Chang, 1977; Carr and Bower, 1980; Bickerstaff, 1982;

Mosbach, 1987). Furthermore, useful information about the primary,

secondary, tertiary and quaternary structures of proteins have been

obtained from a wide variety of researches based on immobilized

enzymes (Woodword and Wiseman, 1978; Bickerstaff, 1980; Amarant

and Bohack, 1981). Enzymatic reaction mechanism (Klemes and Citri,

1979; Askari et al, 1980; Doubradi et al, 1980), intracellular regulation

mechanisms (Swatsgood et al, 1976; Landman and Lampert, 1978) and

several features of multi enzyme systems (Gestrelius et al, 1972;

Mosbach and Mattiason, 1976; Tischer and Kasche, 1999) have also

been elucidated through investigations using immobilized enzymes.

Immobilization often improves the operational stability of enzymes

(Clark, 1994).

18

The last three decades have witnessed the development of large

number of methods and techniques for the immobilization of biologically

active proteins applicable to a variety of enzymes. Universally applicable

techniques are however not available for all enzymes and the choice of

method is decided depending on nature of the enzyme.

The enzyme immobilization methods have been broadly classified

into covalent attachment to or adsorption on solid supports, entrapment

in polymeric gels, encapsulation and cross-linking (Fig. 1.2).

1.3.1 Use of monoclonal/poly clonal antibodies

Antibodies evolved as an important means of defense against

infection. It was therefore reasonable to assume that their relevance

would rest exclusively in this realm, with applications restricted to

determining the infection response and the nature of antigen

involvement. Exploration of other avenues revealed that the antibodies

that offer limitless range of specificities can also be used to define,

isolate and measure an array of immunogenic molecules.

One aspect that has been receiving remarkable attention is the

immobilization of enzymes using antienzyme antibodies, since specific

antibodies can be raised against any enzyme in suitable animals (Ehle

and Horn, 1990). Considerable informations for the immunization,

isolation of specific antibodies and their immobilization for the

immunoaffinity purification and immobilization of enzymes are now

available (Catty and Raykundalin, 1988).

19

Fig. 1.2 Principal modes of enzyme immobilization

20

y^A Adsorption Covalent Binding

-' "

^ >

Encapsulation

Cross-linking

Entrapment

Both polyclonal and monoclonal antibodies have found wide

application in the immobilization of enzymes (Table 1.3). Antiserum of

an immunized animal may contain many different kinds of antibodies

directed against various epitopes of the structurally complex

immunogen. The major advantage of polyclonal antisera lies in their

capacity to form large insoluble immune complexes with antigen.

Polyclonal antibodies are the products of many responding clones of

cells, and hence, heterogenous at many levels in the classes and

subclasses, specificity, titre and affinity. For most enzyme

immobilization application however, heterogeneity of the antibody

population may not cause any serious problem provided they do not

comprise of inhibitory and/or labilizing antibodies. Formation of active

site recognizing and hence inhibitory antibodies is quite likely if an

animal is immunized with a native enzyme (Cinader, 1967; Amon, 1973;

Soloman et al, 1984), although several reports describing the non-

inhibitory nature of the antisera raised against several enzymes are

available (Shami et al, 1991; Jafri et al, 1993). Some ingenious

techniques for prevention of the formation of active site directed

antibodies have also been described in recent years (Fusek et aL, 1988;

Stovickova et aL, 1991).

Monoclonal antibodies exhibit remarkable specificity since they

are directed against a single epitope of the antigen. Hybridomas can

provide a wide spectrum of monoclonal antibodies from which a

continuous supply of appropriate antibody population can be ensured

(Solomon et al, 1984). Monoclonal antibodies that are non inhibitory

and confer remarkable stability to enzymes have been isolated and are

21

M l

-3 o a 93

O

a, J3 0)

,£3 - 4 1 ^

J3 ' 4 1 ^

> ^

2 o >

C M

-o a> ^N

IZS . f i o a s V3 0) s

a V 0

3 e« H

^4-1

05

I f l c '3

c

ca N

r at

*-* 1/5

>< (8

E

o a 3

1/3

>. T3 O

1 N

<1)

6 N C

o Os

c CO

)2 u

2i 3 ca

a E H

1

U a,

•"2 o o _w 13 O

1

ca

0 0 0\

n 0 0 O OS

ca •^- '

ca rf c/3 =2

"o

s " £ 2 S 2 > u

•> E

T3 CB

1) W5 o b« ca

U

u D a. Qu

O 1) c«

0) ea c c

^1 ca j a

o ^

O H

0 0

"3 %1 c o E _o

(/3

0 0 ON

"3

c o E _o C/3

2 la O .

E <a H

U .- 0 0

^ u 3

w

u

<

ca

o.

o X I ca U

0 0 . _

Si 00 C ^ 0 0 o\

w "3 c » O li)

E M

O 3 on CL,

2 "o U c • ^ 0 0 &o ca ca o o c/5 55 1

U u •-< o bO !-• ^ ca a j= & n. 3 (U W on

U U

ca 3 u 0 0

o « • 3 ^ >^2

S 8 ^ 5

OS 0 0 OS

"5 *-* ^ E ca

y o a.

j s

E _ 3

•3 o C/5

1

U a.

c

3 on

OS 0 0 OS T — 1

"5 .^ cu

O 3

a5

c _o ca

O . O

b- ^ b« 3 3 3

ca ca ca kri V« ^

a> cu o 0 . 0 . 0 . E E E u :^ •u E

* - . ca

2 J= is o.

6 0

c ca

6 0 _C

'N

fe

T-H OS 0 \

^ a

E ca

Os" 0 0 0 \ r H

"a 'S E ca

C/5

"o c ca

W

2 « b -

o. E H

1

U CL,

u

o OS OS

a 'S c o E

_o "o en

1

1

U

0) CA ca

o

o. CA r -

E s ca "-o u O CA

O X

^—V r H OS OS

53

ca > o M

u '> o

on

1

(L> lA

o w. ca

j i O .

c/5

U

'CA O . >>

CA

o ^^ ^^ >-. ( S rsi - ; : OS OS O OS OS T^ ^ H / V ~ ^ ^-' cn

r r OS " S " S OS

p 'S «

3 C ' C 6 0 6 0 « < < 5

3

ca 0) ^

X 2 S o.c/3 H

u CA

o k .

c c ca _ 0 O 4 =

• 5 ^ k i ^ "1> 2 Z 00

'o U U U o. O^ O H CU 1/3

0) CA

ja i- i

O u u « > . ca

c^ ^% "^ ca Q > H < = D Z

OS

_c •3 -a 3

E ID

1/3

T3 3 ca

ca • * • -1 OS

-. OS

^ ^ OS OS ^ ^

- J " a c «- c (u ca •c E —1 1/3

3 3

« .2 a> ca O . X I

6 3

u u CA CA

o o k- U<

ca ca s: j : o. o.

y 3 1/3

U U

ca 3

'S 'E ca

T3

1

ca 3 _o o B O

u "ca c

cu 1

U CU OS

OS C3S

-a 3 E

C/3

3 O

C/3

being used increasingly in the immobilization of enzymes (for review

see Turkova, 1993; and Saleemuddin, 1999). Culturing of hybridomas

however remains an expensive and time consuming endeavour.

1.3.2 Enzyme immobilization with the help of antibodies

A variety of approaches have been adopted for the immobilization

of enzymes with the help of antibodies. The earliest being the

preparation of enzyme antibody complex for which neither pure antibody

nor pure enzyme is a prerequisite. Burnett and Schmidt (1921)

demonstrated for the first time that the insoluble immunocomplex of

catalase and anti catalase antibodies retained complete catalase activity.

Other workers reported high retention of catalytic activity and marked

stability against various denaturing factors after enzyme antibody

complex formation (Campbell and Fourt, 1939; Shami et al, 1989;

Shami et al, 1991; Jafri and Saleemuddin, 1997). Micheali et al. (1969)

prepared acetylcholine anti-acetlylcholinesterase complexes with high

enzyme activity and enhanced resistance against thermal denaturation at

60°C. Sato and Walton (1983) improved the immobilization and

stablization of gluconolactone oxidase using polyclonal antibodies raised

in guinea pigs.

Use of antigen antibody interaction for affinity binding of proteins

and enzymes is a highly selective procedure, widely used in

immunoaffinity chromatography. The procedure involves in the

attachment of antibodies to solid supports like agarose, Sepharose or

synthetic organic and inorganic porous bead matrices (Gronman and

23

Wilcheck, 1987), The most widely used immobilization technique

involves the reaction of nucleophilic group (mostly primary amines) of

the antibody with polysaccharide support activated by CNBr (Coding,

1983), epoxy activation using epichlorohydrin (Pepper, 1992),

bisoxiranes (Sunderberg and Porath, 1974) or by tosyl/tresylchloride

activation (Nilsson and Mosbach, 1980; Nilsson and Mosbach, 1981).

Zusaman and Zusaman (1995) immobilized IgG on a support prepared

from glass fibers covered with oxysilanes to get a gel fiber-glass matrix.

Bilkova (1999) showed that immunoaffinity matrix prepared for

chymotrypsin immobilization retained practically 100% of its native

activity with improved kinetic parameters. Ikura et A/. (1984) observed

higher activity of guinea pig liver transglutaminase or immobilization

using specific monoclonal antibodies compared to the matrix precoupled

with the polyclonal antibodies. The immobilization exhibited improved

thermal stability.

An effective way of immobilizing glycoenzymes involves the

binding to antibody supports that specifically recognizes the glycosyl

residues of the protein. As the enzyme glycosyl residues rarely

participate in the catalytic activity (Lis and Sharon, 1993), glycosyl

recognizing antibody should normally be non-inhibitory and this can

yield active enzyme antibody complex. Strategies have been designed to

obtain polyclonal antibodies directed against glycosyl groups in

glycoproteins and glycoenzymes (Zopf et al, 1978; Feizi and Childs,

1990; Jafri et a/., 1995). It was also reported that glycosyl recognizing

polyclonal anti invertase antibodies were non-inhibitory and yielded

superior immobilized enzyme preparations compared to those

24

recognizing the polypeptide domains (Jafri et al, 1995; Jafri and

Saleemuddin, 1997).

1.3.2.1 Oriented immobilization of antibodies

Most of these immobilization procedures lead to a random

immobilization of the antibody molecule on the support that may result

in steric hindrance to the access of the antigen binding site and in

reduced binding activity. Several attempts to favourably orient

antibodies on support matrices have therefore, been made.

Immobilization of the antibody on an immobilized protein A matrix for

instance leads to its favourable orientation (Gersten and Marchalonis,

1978; Solomen et al, 1986). This is related to the ability of the protein

A to bind to the Fc region of IgG. Reports are also available for the

oriented immobilization of antibodies on various supports via their

oligosaccharide chains, which are mainly located in the Fc region of the

molecule (Quash et al, 1978; Hoffmann and O'Shannessy, 1988;

Fleminger et al, 1990). More recently, Bilkova et al (1999) prepared

enzymatically active bioaffinity matrix with suitable polyclonal

antibodies for oriented immobilization of chymotrypsin. For this purpose

nonporous microspheres were prepared in alcohol toulene mixture

stabilized with cellulose acetate butyrate. Reactive groups available after

modification with adipic acid were used for coupling of

antichymotrypsin antibodies. Attempts have also been made to orient

antibodies through their -SH groups of the Fab fragment at the C-

terminus (Prisyazhony et al, 1988).

25

1.3.2.1.1 Immobilization on metal ion affinity support

Immobilized metal ion affinity chromatography (IMAC) is a

highly versatile separation technique based on interfacial interactions

between metal ions immobilized on a solid support, which is usually a

hydrophilic cross-linked polymer and basic groups on proteins, mainly

histidine residues. But sometimes terminal amino group, tryptophan and

cysteine residues may also be involved. The concept of using chelated

metals for the selective adsorption of proteins was first introduced by

Porath et al. (1975). Since then IMAC has found several applications

including those in the separation of red blood cells (Sulkowiski, 1989)

and recombinant E. coli cells containing surface hexahistidine clusters

(Sousa et al, 1996).

First row transition metals (Ni , Zn^^, Cd^, Cu^ , Fe^ ) are

generally used as ligand after chelation by iminodiacetic acid (IDA).

IDA is a trident chelator that binds metal ions through its nitrogen and

two carboxylate oxygens as seen from Fig. 1.3. As the metal ion

coordinates 4-6 ligands, the remaining of coordination sites are occupied

by the water molecules and buffer ligands which are available for

replacement by appropriate surface groups (Arnold, 1991).

The potential of IMA in enzyme immobilization is being

increasingly realized. Several proteins and enzymes have innate affinity

in their native state for the metal chelate support due to the presence of

one or more surface histidine and bind to metal affinity supports

according to number of available histidine residues (Hemdan et al,

26

Fig. 1.3 Principle of enzyme immobilization by metal-chelate

bonding with regeneration of the carrier

27

Jvwy NH^gg l^^^gg: Me- I

o

<-x H,C.

g' | vwy

'Jatrix Octane activated garose) niatrix

NaOH

Regeno^on (e.g.EDTA)

x-HjO or con]]dexing ion

IDA gel i

metal-chelate gel

1989). Coulet et al (1981) immobilized lactate dehydrogenase, malate

dehydrogenase and alkaline phosphatase on Co^*, Zn ^ and Cu^ -

chelating Sepharose. All the enzymes were active in the immobilized

state although the specific activity was quite low. A number of other

proteins and enzymes are also known to bind strongly to the metal

chelate supports including ribonuclease A and lysozyme (El Rassi et al,

1988).

Immobilized metal chelate supports have also been used in the

preparation of affinity sorbents for enzymes that do not exhibit high

affinity for the immobilized metal ions. This has been achieved for

instance with the help of proteins that strongly bind both to the carriers

and to the enzymes. Concanavalin A (Con A) binds strongly to Cu ^

iminodiacetic acid functions in view of the presence of six histidine

residues in each subunit (McKenzie et al, 1972) and retains affinity

towards glucose and mannbse as well as glycoproteins bearing the

residues. Infact, Con A appears to be oriented on immobilized metal ion

support in a manner that facilitate high accessibility towards the

glycosyls (Anspach and Altmann, 1994). Con A immobilized on metal

chelate has been shown to bind a number of enzymes including

peroxidase and glucose oxidase (El Rassi et al, 1988).

Hale and Beidler (1994) demonstrated that antibodies bind to

Co^*-IDA-support via metal binding sites located on C-terminal portion

of the heavy chain. This facilitated the orientation of the antibody with

antigen binding site facing away from the support. It was also possible to

immobilize antibodies in an exchange inert fashion by oxidizing the

antibody bound Co^* to Co^* and all the investigated subclasses of the

28

antibodies could be immobilized by this procedure (Hale, 1995).

Considerable potential of enzymes immobilized on immunoaffinity

support exist in industry and analysis (Turkova, 1993) and the Co "

chelate supports may be useful in preparing supports with favourably

oriented antibodies for the purpose.

1.3.3 Immobilization of Restriction Enzymes

The physico-chemical properties of REs in relation to their

activity and stability have been discussed in recent reviews (Dubey et

al, 1987; Rosenberg, 1991). The usually low stability and high cost of

production of highly active and specific enzymes in native form has

limited their applications in biotechnology. Use of immobilized REs

should be certainly advantageous in the cloning technology, as it allows

their easy removal from the reaction mixture as well as their reuse.

These procedural advantages together with potential enhancement in

stability clearly make the insoluble form of these enzymes more valuable

(Tietz, 1980; Wiesman, 1980). Taking these into considerations some

attempts have been made to prepare immobilized preparations of several

restriction endonucleases.

Immobilized EcoRl and BamYH prepared by covalently coupling

them to CNBr-activated Sepharose, show significant enhancement in

thermal stability with retention of 50-90% activity (Lee et al., 1978). In

addition to high operational and storage stabilities the insoluble

endonucleases cleaved X, adenovirus and SV40 DNAs with the

specificity of soluble enzyme and the preparations could also be

29

lyophilized without the loss of activity. Lee et al (1980) subjected

activated cotton to periodate oxidation and the generated aldehyde

groups thus were used for the coupling of BamWl. No changes were

observed when the action patterns of both soluble and BamWl

immobilized thus were compared on several viral DNAs. In attempts to

immobilize EcoRl, Woodhead and Malcolm (1981) covalently coupled

carbodimide-metho-sulphonate to side chain carboxyl group of EcoKl

but the treatment inactivated the enzyme.

Side chain amino groups of Pst\ cannot be used for coupling of

the enzyme to solid support (Singh, 1984). This was inferred from sets of

experiments in which acetylation of amino groups of Pstl with

hydroxysuccinamide acetate or binding of the enzyme through its amino

group to CNBr-activated Sepharose resulted in the loss of catalytic

activity. In a related study, Nasri and Thomas (1987) assessed the

influence of various amino acid modifying reagents to determine the

functional groups involved in the catalytic activity of PVMII and HindlU.

Treatment with glutaraldehyde or carbodimide inactivated both the

enzymes and the inactivation could not be prevented by inclusion of

pBR322 DNA in the reaction mixture. On the contrary, Mg"* protected

the enzymes against inactivation, suggesting the involvement of

carboxylate groups in metal binding. Glutaraldehyde mediated

inactivation, on the other hand, was attributed to the modification of

lysine residues rather than conformational changes, as lysine

modification by methyl acetimidate also resulted in loss of enzyme

activity. While N-ethylmaleamide caused slight inhibition of Pvull, it

had no effect on the activity of HindlW indicating that cysteine may not

30

have direct role in the catalytic activity of the later. PVMII and Hindlll

when immobilized through side chain amino or carboxylic group did not

show any detectable activity. Moreover, they retain partial activity when

immobilized on pAB cellulose through phenolic groups. The decrease in

the activity of immobilized enzyme was attributed to the modification of

functional groups at or near the active site, in addition to the resulting

steric and/or diffusional limitations (Porath et al, 1961 \ Axen and

Emback, 1971). Olszewski and Wasserman (1986) made systematic

investigations on the effect of glutaraldehyde on the activity of

restriction enzymes Bam\i\, Hindlll, £'coRI and Tthlll. Glutaraldehyde

treatment was carried out in the presence of HEPES, MOPS or

phosphate buffer, because primary amino group in Tris can severely

interfere with the reaction. Tthlll was the most sensitive with activity

losses occurring at very low concentrations of glutaraldehyde, and

Hindlll the most stable. Because the glutaraldehyde reaction primarily

involves the £-amino groups of lysine (Richards and Knowles, 1968), the

sensitivity to glutaraldehyde points towards the probable involvement of

lysine in the catalytic activity of some of these enzymes and/or

conformational changes as a result of cross-linking.

Enzymes immobilized on water insoluble polymers may lose their

capacity to react with water insoluble substrates and may not be fully

accessible to large substrates. In order to overcome the possible steric

hindrance of the matrix, Dubey et al. (1989) attempted to stabilize

BamHl by cross-linking the enzyme with various bifunctional agents viz

glutaraldehyde, dimethyl adipimidate (DMA), dimethylsubermidate

(DMS) and dimethyl 3,3'-dithiobispropionimidate (DTBP). The DTBP

31

cross-linked BamHl was the best heat stable preparation among those

tested as compared to DMA and DMS treated preparations.

Cashion et al (1982a) attached EcoRl, BamHl, Hindlll, DNA

polymerases, RNA ligase, SI nuclease, AMV reverse transcriptase etc.

via hydrophobic interactions to tritylagarose or Sepharose with high

retention of activity. They also immobilized EcoRl, BamHl and Hindlll

on tritylated agarose with partial retention of the respective enzyme

activities (Cashion et al, 1982b) only for five to twelve days. It was

further demonstrated that because of the non-covalent association

between enzyme and support, both could be reused.

Singh (1984) also entrapped Pstl in a low temperature melting

agarose in the presence of 1, 6-hexane methlylenediamine and the

preparation effectively linearized pBR322 DNA. Entrapment in

polyacrylamide resulted in a considerable reduction in the activity of

Pstl, whereas £coRI under identical conditions exhibited high activity.

The low activity of polyacrylamide entrapped Pstl was attributed to

inactivation of the enzyme rather than to diffusional barriers because

entrapped EcoKl showed high activity under the conditions. The

problem of diffusional limitations of polymer entrapped enzymes to large

substrates prompted Wingard and Egler (1984) to couple lysine to

several restriction endonucleases including EcoKl, BamHl and Bgll,

using water soluble carbodimide for attaching these enzymes through

their carboxylate groups to solid supports. Because coupling of lysine to

these enzymes does not affect their catalytic activity, it was concluded

that binding via carboxylate groups may be useful for immobilizing the

restriction endonuclease.

32

It was of interest to note that the alteration in endonuclease

specificity of soluble Pvull and Hindlll occurred when cleavage reaction

was carried out in presence of dimethyl sulphoxide (DMSO), though no

change in activity was observed with immobilized endonucleases. This

loss of specificity of soluble enzyme was correlated with the

conformational changes of the active form of the enzymes and the

absence alteration in case of the bound enzyme to the rigidity of the

three dimensional structure of the immobilized enzyme (McLaren and

Packer, 1970).

In a recent sending Bircakova et al. (1996) attempted the oriented

immobilization of EcoKl on Sephadex G-10 by linking it via its single

free sulphydryl group. For comparison EcoKl randomly coupled via the

side chain carbonyl groups was used. The superiority of the proposed

oriented enzyme was demonstrated in terms of its action on low and high

molecular weight substrates, storage stability and reuse.

33

Experimental

2.1 Materials

2.1.1 Chemicals

Chemicals used for the studies were obtained from sources given

against their names. Glass distilled water was used in all the

experiments.

Source Chemicals

B.D.H., India copper sulphate, ethanolamine

B.D.H., Poole, England bromophenol blue, sucrose

Difco Lab., USA

E, Merk, Germany

E. Merk, India

Genei Laboratories, India

diethyaminoethyl cellulose, freund's complete adjuvant, freund's incomplete adjuvant.

P-Mercaptoethanol

acetonitrile

EcoRl, BamEl, pBR322, pUC19, k DNA, nuclease free BSA, lysozyme

Hi-Media, India agar agar, nutrient broth, ampicillin, tetracycline

Qualigens Fine Chemicals, India

acetic acid, calcium chloride, cobalt chloride, di-potassium hydrogen phosphate, isopropanol, methanol, potassium phosphate, sodium carbonate, sodium chloride, sodium phosphate, sodium potassium tartarate, triton X-100, potassium chloride, magnesium chloride.

Sigma Chemical Co.,USA agarose, coomassie brilliant blue R-250, human

serum albumin, N, N, N', N', tetramethylene diamine, sepharose 4B, sodium lauryl sulphate,

34

tris (hydroxy methylamino methane),

^. _. , T 1 acrylamide, ammonium per sulphate, ammonmm Sisco Research Lab., / , ' . ^ „ . , ,. sulphate, bovme serum albumm, cyanogen

bromide, dithiothreitol, ethylene diamine tetra acetic acid, folin's reagent, formaldehyde, glycerol, glycine, N', N', methylene bis acrylamide

2.1.2 Bacterial Strains

Prof. H. K. Das of Jawaharlal Nehru University, New Delhi, India,

kindly provided a strain E. coli pMB4 that overproduced EcoRl. In this

strain EcoRl is coded by a multicopy plasmid and therefore the enzyme

present is in much high concentration. Another recombinant strain E. coli

JM109 (3P) was received from K. Schuegerl and J.I Rhee, of Institute of

the Technical Chemistry University of Hanover, Germany. E.coli JM 109

(3P) carries the combinations of three plasmids -expression plasmid

pMTC48 (4.8 kbp) carrying the gene SpA::£coRI under the

transcriptional control of two independent promoters, P|acuv5 promoter

inducible with isopropyl thiogalactoside (IPTG) and thermally inducible X

PR promoter. In addition it carries the ampicillin resistance gene. The

repression plasmid pRK248 (9.2 kbp) overproduces the cI857 repressor

protein and carries the tetracycline resistance gene. The protection

plasmid pEcoR4 (6 kbp) encodes the DNA methylase of EcoRl and

carries the chloremphenicol resistance gene. For more details, see

Maschke et al. (1992).

35

2.1.3 Media

a. Luria Bertani, per litre -10 g casein peptone-, 5 g yeast extract, 10

g sodium chloride.

b. Nutrient Broth, per litre - 5 g peptone, 5 g sodium chloride, 1.5 g

yeast extract, 1.5 g Beef extract, 15 g Agar, Final pH (at 25°C) 7.4 ± 0.2.

c. M9 Minimal medium* (10 X), per litre-

100 ml salt solution: 21 g Na2HP04, 25 g KH2P04_ 5g NaCl, 25g

(NH4)2S04,

1 ml 0.1 M CaClj, 1 ml 0.1 M MgCl2.

100 g glucose solution.

Salt solution, glucose solution, MgCl2/CaCl2 solutions were autoclaved

separately.

2.2 Methods

2.2.1 Cell Cultures and Preparation of Lysates

2.2.1.1 Cultivation ofE. coli pMB4

E. coli pMB4 was grown in seven ml of 13 g/100 ml nutrient

broth (NB) containing 10 |xg/ml ampicillin in sterilized tubes overnight at

37°C. One hundred \i\ of the cell suspension was transferred from N.B

medium to 200 ml conical flasks having 100 ml L.B medium with 10

mg/ml ampicillin added after autoclaving. The medium was cooled and

36

incubated with gentle agitation at 37°C in a shaking water bath overnight.

The cells were pelleted down by centrifugation.

2.2.1.1.1 Cell lysis

Cells lysis was achieved by chemical-enzymatic method with

EDTA/Tris and lysozyme (Pleczar et al, 1986, Wiseman, 1969). Pelleted

cells were suspended in minimal volume of TA buffer (50 mM Tris-HCl

pH 7.6, 30 mM NaCl) then equal amount of lysis buffer (50 mM Tris-HCl

pH 7.6, 30 mM NaCl, 12 mM EDTA, 400 (xg/ml lysozyme, and 40%

sucrose) was added to cell suspension and the mixture was stored at 0°C

overnight. The cell debris and the unlysed cells were removed by

centrifugation at 9800 x g for 30 min at 4°C and the clear suspension was

used as crude homogenate for immobilization of ^coRI.

2.2.1.2 Cultivation oiE. coft JM109

In slab culture the strain were first grown in nutrient broth

overnight under selection pressure of antibiotic tetracycline at a

concentration of 10 mg/1, (Maschke et al, 1992). One hundred il of the

turbid suspension of NB medium was streaked on agar plates having LB

medium with 17g/l agar supplement. The medium was autoclaved and

poured into a petridish at 50-55°C and incubated after streaking at 30° C

overnight. The cells were collected from the petriplates and transferred in

200 ml of M9 medium in 500 ml conical flasks at 30°C. One ml of the

M9 culture was transferred and cells were cultivated in 200 ml LB

37

medium at 30°C for 18 hours. The cultivated medium was supplemented

with 1/20 or 1/10 volume of 20X or lOX concentration LB medium and 5

g/1 glucose and the temperature was raised from 30°C to 40°C to induce

the P^-Promoter (Rhee et ah, 1997). After three hours incubation the cells

were pelleted by centrifugation at 9800 x g at 4°C for 30 min. The cells

were lysed with the chemical-enzymatic methods with EDTA/Tris and

lysozyme (Pleczar et al; 1986, Wiseman, 1969). For this purpose the

cells were mixed with one volume of lysis buffer (400 ^ig/ml lysozyme,

30 mM NaCl, 12 mM EDTA, 40% sucrose) and one volume of TA buffer

(50 mM Tris-HCl pH 7.6, 30 mM NaCl) at 0°C overnight.

The cell debris and unlysed cells were removed by pelleting at

9800 X g and 4°C for 30 min. and the clear supernatant was used as a

source ofEcoRl- protein A.

2.2.1.2.1 Preparation and fractionation of crude bacterial lysate

In the small scale experiments the E. coli JM109 (3P) cell pellets

were suspended in 0.05 M Tris-HCl (pH 7.6), 10% sucrose, 2 mM DTT

and 5 mg/ml lysozyme and kept overnight for lysis at 0°C. After lysis the

cell extract was centrifuged to remove the cell debris. The crude extract

obtained thus was fractionated by freshly prepared 25% (w/v)

streptomycin sulphate. After stirring at 0°C for 45 min, the suspension

was centrifuged at 9800 x g for 20 min. and the supernatant collected.

Solid ammonium sulphate was added to 25% saturation, the

precipitate collected by centrifugation and the supernatant was again

made 50% saturation with ammonium sulphate. The precipitate was

collected and dissolved in 0.02 M potassium phosphate buffer pH 7.4

containing 1 mM EDTA, 5 mM p-mercaptoethanol, and dialysed against

38

0.02 M potassium phosphate buffer pH 7.4, containing 5 mM 2-

mercaptoethanol, 10% w/v glycerol with 0.5 mM EDTA. These dialysed

samples were further used as the source of £'coRI

2.2.2 Immunological Procedures

2.2.2.1 Preparation of the antigens

For raising the anti EcoRl antibodies in rabbits, 95 xl of EcoRl (400

U/100 \i\ of storage buffer containing 10 mM Tris-HCl, pH 7.4, 200 mM

NaCl, 0.1 mM EDTA, 7 mM |3-mercaptoethanol, 50% glycerol, 200

ig/ml nuclease free BSA and 0.2% Triton X-100) was mixed with 300 il

of sterilized distilled water and thoroughly emulsified after adding 100 \i\

of Freund's complete adjuvant using a 21 gauge syringe. Booster doses of

antigens were prepared by mixing 400 |xg of EcoRl with 300 yd of

sterilized distilled water and emulsified with 100 yd of Freund's

incomplete adjuvant.

Antigen for immunizing rabbits against BamHl antibodies was

prepared by mixing four hundred microgram of BamHl supplied

commercially in storage buffer having 10 mM Tris-HCl (pH 7.4), 100

mM KCl, 0.1 mM EDTA, 7 mM p-mercaptoethanol, 50% glycerol, 200

[ig/m\ nuclease free BSA and 0.05% Triton X-100 with 200 il of

sterilized distilled water along with 100 il of Freund's complete adjuvant

Antigen for the booster doses was prepared similarly but using the

Freund's incomplete adjuvant.

39

2.2.2.2 Immunization

Healthy albino rabbits weighing between 1.5-2.0 kg were selected

for immunization. The rabbits were bled prior to immunization and the

sera obtained served as control.

Rabbits were injected subcutaneously at multiple sites with 0.5 ml

of EcoRl or BamHl antigens. Animals received 400 ig of antigen along

with the adjuvant weekly over a period of four weeks. The animals were

rested for two weeks and administered four weekly booster doses of 400

pig of emulsified enzyme in case of EcoRl and six weekly doses for

BamHl. After further resting of the animal for a week, blood was drawn

by cardiac puncture and clot formation allowed at room temperature for

two hours. The blood was centrifuged to collect the antiserum, which was

then decomplemented by heating at 56°C for 30 min, mixed with sodium

azide, quickly frozen and stored in small aliquots at -20°C till further use.

2.2.2.3 Oucterlony double diffusion

The procedure of immunodiffusion as described by Oucterlony

(1949) was used to study the precipitin reaction between antigens and

antibodies. Initially 30 ml of 1% agarose solution prepared in normal

saline was boiled to dissolve the agarose and poured on to the glass plates

and allowed to set. After the solidification of the gels, wells were

punctured in the centre and periphery. Twenty-eight microliters of

antiserum was loaded in each peripheral well while 28 pil of antigen

solution in the central well, which was added after eight hours of

application of antiserum. The plates were incubated at room temperature

overnight for the reaction to proceed.

40

2.12A Isolation and purification of IgG from the antisera

2.2.2.4.1 Ammonium sulphate fractionation

The antisera raised against the EcoRl or BamHl were fractionated

in order to obtain the IgG fraction. Twenty ml of antiserum was mixed

with half the volume of 20 mM sodium phosphate buffer and 3.42 g of

the solid ammonium sulphate. The mixture was stirred to dissolve

ammonium sulphate and to make it 20% saturated. After 6 h the sample

was centrifuged at 5000 rpm for 20 min and the supernatant was made

40% saturated with respect to ammonium sulphate by adding 3.85 g of

the solid. The precipitate obtained after 16 h at 4°C was collected by

centrifugation at 5000 rpm for 20 min. Most of the gamma globulin

fraction was present only in the 20-40% pellet that was dissolved in

minimal volume of 20 mM sodium phosphate buffer, pH 7.2, and

extensively dialysed against same buffer with several changes in order to

remove the ammonium sulphate. This partially purified anti EcoRl IgG

fraction was used for the immobilization on activated Sepharose 4B

while, anti BamHl immunoglobulin fraction was further purified by ion

exchange chromatography.

2.2.2.4.2 DEAE-cellulose chromatography

Ion exchange chromatography of the 20-40% ammonium sulphate

fraction was performed using DEAE-cellulose (Fahey and Terry, 1979).

Required amount of DEAE-cellulose was suspended in 10 volumes of 0.5

N HCl for 1 h at room temperature and washed in a Buchner funnel with

distilled water till the filtrate had neutral pH. The exchanger was then

treated with 0.5 N NaOH for 1 h and again washed extensively with

distilled water in order to remove excess of base. The regenerated DEAE-

41

cellulose was then resuspended in 20 mM sodium phosphate buffer, pH

7.2 to obtain homogeneous slurry. Fine particles were removed by

decantation and the slurry poured in a clean vertically mounted column

(2.0 X 10 cm). The flow rate was gradually increased to 0.5 ml/min, the

buffer carefully removed from the surface and the dialysed ammonium

sulphate fraction was applied. Three ml fractions were collected after

washing the column with one bed volume of 20 mM sodium phosphate

buffer pH 7.2 and the fractions analysed for protein content.

Goat IgG was purified in the similar manner to that of rabbit IgG as

described except that 30 mM phosphate buffer was used.

2.2.3 Immobilization of Antigen/Antibody

2.2.3.1 Immobilization of IgG on Sepharose 4B

Sepharose 4B was activated as described by Porath et al. (1967).

Two g of Sepharose was washed thoroughly in a Buchner funnel with

distilled water. The gel was sucked dry and then suspended in 2 ml of

distilled water and 2 ml of 2.0 M Na2C03. The slurry was stirred at room

temperature for 15 min by placing it on a magnetic stirrer.

Four hundred milligrams of CNBr dissolved in 400 il of

acetonitrile was added to the beaker containing Sepharose and mixed

thoroughly in cold for 10 min. The whole mass was rapidly transferred to

a glass-sintered funnel and washed successively with excess 0.1 M

sodium bicarbonate buffer, pH 8.5, distilled water and again with the

buffer.

Appropriate amount of partially purified or purified anti £'coRI/anti

BamWl IgG fraction was added to the CNBr activated Sepharose. The

suspension was stirred gently for 24 h at 4°C. The Sepharose bound

42

protein was separated by centrifugation and the protein in the supernatant

quantitated by the procedure of Lowry et al. (1951). In order to calculate

the amount of protein bound. The amount of protein in the supernatant

and combined washings was subtracted from that added. The protein

bound matrix was thoroughly washed with 0.1 M bicarbonate buffer pH

8.5 containing 1.0 M NaCl and subsequently with 0.1 M bicarbonate

buffer pH 8.5 alone.

The washed suspension was then treated with 0.1 ml of 98% (w/v)

ethanolamine for two hours at 4°C. The matrix washed with 0.1 M

bicarbonate buffer pH 8.5 containing 1.0 M NaCl, distilled water and

finally with 10 mM potassium phosphate buffer, pH 7.4 and stored in the

same buffer.

Goat IgG was immobilized on CNBr activated Sepharose exactly as

described for rabbit IgG except for the replacement of 20 mM phosphate

buffer with that of 30 mM.

2.2.3.2 Immobilization of liuman serum albumin on CNBr activated

Sepharose 4B

HSA Sepharose was prepared according to the procedure of

Porath et al. (1967), as described above to obtain a preparation containing

18 mg HSA coupled per ml gel.

2.2.3.3 Oriented Immoblization of Antibodies

2.2.3.3.1 Immobilization of anti BaniW. IgG on metal affinity support

For the immobilization of anti BamYil immunoglobulin G fraction

on metal chelated IDA Sepharose, the procedure described by Hale

(1995) was followed with slight modification. Five hundred microliters of

43

fast flow iminodiacetic acid (IDA) Sepharose was washed with 50 mM

EDTA, 500 mM NaCl and finally with distilled water. The washed IDA

resin was shaken end to end with 2.0 M CoClj solution for 30 min.

Unbound metal was washed of with excess distilled water. The Co^

bound matrix obtained thus was equilibrated with 0.1 M sodium

phosphate buffer pH 7.5. Purified IgG (5 mg/ml) was allowed to bind on

the matrix for about 4 h in an eppendorf tube after which the supernatant

containing unbound protein was collected, washed repeatedly with

phosphate buffered saline (PBS), with distilled water and again with PBS.

The IgG-Co^*-IDA-Sepharose obtained thus contained 1.7 mg/0.5 ml

bound IgG. The support with bound cobalt and anti BamRl IgG was

incubated with 0.3% H2O2 in PBS for 2 h at room temperature. After

oxidation the support was washed with PBS followed by the buffer

containing 50 mM EDTA, 0.5 M imidazole and finally stored in PBS.

The resulting preparation was used as immunoaffinity support for the

immobilization ofBamHl.

2.2.3.3.2 Immobilization of anti HSA IgG

HSA-Sepharose was prepared according to the procedure

described by Porath et al. (1967). For the oriented immobilization of IgG,

Sepharose support with 18 mg bound HSA/ml gel was used. One ml of

HSA Sepharose washed with 20 mM sodium phosphate buffer, pH 7.2

and incubated with required amount of IgG in the same buffer for 16 h at

4°C. The IgG coupled Sepharose was centrifuged at 3000 rpm for 5 min

in order to remove the unbound IgG. The matrix was washed repeatedly

with 5 volume of same buffer in order to remove loosely bound IgG. The

amount of protein immobilized was calculated by subtracting the amount

44

in the supernatant and the first washing from that added to the support.

The preparation obtained thus was used as affinity support to bind

SpA::£'coRI through Fc portion of IgG bound to HSA Sepharose.

2.2.4 Enzyme Immobilization

2.2.4.1 Immobilization of EcoRl/BamHl on IgG Sepharose

The enzymes were immobilized by incubating 200 units of ^coRI

or BamEl with 250 il of Sepharose precoupled with respective

antienzyme IgGs. The support was pre washed at least 4-5 times with 10

mM potassium phosphate buffer containing 0.5 mM EDTA, 50% glycerol

and suspended in 500 yd of the same buffer. The whole suspension was

gently mixed 'end over end' at 4°C for at least 5 h. The excess unbound

endonuclease was removed from the gel by centrifugation, resuspended in

0.5 M NaCl, 25 mM Tris-HCl pH 7.5, 10% glycerol, and ImM

dithiothreitol followed by recentrifugation. This washing procedure was

repeated four times. Finally, the enzyme-coupled gel was equilibrated

with 50 mM Tris-HCl pH 7.5, 20% glycerol, and 1 mM dithiothreitol by

washing five times in the same buffer. The final gel slurry was stored at

4°C.

2.2.4.2 Immobilization ofBamKl on IgG-Co^*-IDA Sepharose

For the immobilization of BamHl on metal chelated support, 250

units of BamHl in the storage buffer containing 10 mM Tris-HCl pH 7.5,

100 mM KCl, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 50% glycerol,

200 fig/ml nuclease free BSA and 0.05% Triton X-100 was incubated

with 250 fxl of IDA-Co^^-IgG resin washed with 10 mM potassium

45

phosphate buffer pH 7.5, containing 50% glycerol and 0.5 mM EDTA at

4°C. The suspension was gently mixed for at least 8 h. The matrix was

then separated from the unbound endonuclease by centrifugation and

washed at least four times with the buffer solution of 0.5 mM NaCl, 25

mM Tris-HCl pH 7.5, 10% glycerol and 1 mM DTT. The enzyme bound

matrix was finally washed thoroughly with 50 mM Tris-HCl pH 7.5, 50%

glycerol, 1.0 mM DTT and stored in the same buffer at 4°C till further

use.

2.2.4.3 Immobilization ofEcoRi from bacterial lysate

For the direct immobilization of EcoRl from the crude lysate, 250

\i\ of immunoaffinity support bearing 19 mg/ml bound anti EcoRl IgG

prepared as described earlier in methods was mixed with 500 jil of E. coli

pMB4 lysate and incubated overnight at 4°C. The support matrix was

separated by centrifugation and washed with 25 mM Tris-HCl pH 7.5, 0.5

N NaCl, 10% glycerol and 1 mM DTT, several times. The enzyme bound

matrix was equilibrated with 50 mM Tris-HCl pH 7.5, 20% glycerol, 1

mM DTT, by washing five times in this buffer. The final gel slurry was

stored at 4°C and suitable aliquots were assayed as described earlier

2.2.4.4 Affinity Immobilization of SpA::£coRI

2.2.4.4.1 Immobilization on goat IgG -Sepharose 4B

Goat IgG Sepharose prepared as described earlier was washed with

10 mM potassium phosphate buffer pH 7.5 containing 0.5 mM EDTA

and 50% glycerol. The preparation containing 19.1 mg IgG per ml gel

was used for the immobilization of the hybrid protein SpA::£coRI. Two

hundred and fifty \i\ of immunoaffinity support was mixed with 1.2 ml of

46

25-50% ammonium sulphate fraction of bacterial lysate and incubated

overnight at 4°C with gentle continuous shaking. The matrix bound

enzyme was separated by centrifugation at 3000 rpm for 5 min. The

Sepharose support with bound enzyme was washed with 10 volumes of

buffer containing 25 mM Tris-HCl pH 7.5, 0.5 N NaCl, 10% glycerol,

and ImM DTT, in order to remove the non specifically bound protein.

The enzyme bound matrix was then equilibrated with 50 mM Tris-HCl

pH 7.5, 20% glycerol, 1 mM DTT, subsequent to washing with 10

volumes of the same buffer. The final gel slurry was stored at 4° C and

suitable aliquots were assayed for EcoRl activity as described earlier.

2.2.4.4.2 Immobilization on favourably oriented IgG.

One ml of Sepharose with anti HSA IgG precoupled to HSA-

Sepharose at a concentration of 18 mg/ml gel was washed with 10 mM

potassium phosphate buffer pH 7.5 containing 0.5 mM EDTA, 50%

glycerol. The preparation containing 19.1 mg IgG per ml gel was used for

the immobilization of hybrid protein SpA::£coRI. Two hundred and fifty

microliters of immunoaffinity support was mixed with excess of 50%

ammonium sulphate fraction of bacterial lysate and incubated overnight at

4° C with continuous gentle shaking. The matrix bound enzyme was

separated by centrifugation at 3000 rpm for 5 min. The Sepharose support

with bound enzyme was washed with 10 volumes of buffer containing 25

mM Tris-HCl pH 7.5, 0.5 M NaCl, 10% glycerol, and ImM DTT, in

order to remove the non specifically bound protein. The enzyme bound

matrix was then equilibrated with 50 mM Tris-HCl pH 7.5, 20% glycerol,

ImM DTT, by washing with 10 volumes of the same buffer. The final gel

47

slurry was stored at 4°C and suitable aliquots were assayed for EcoRl

activity as described earlier.

2.2.5 Estimation of Proteins

The procedure described by Lowry et al. (1951) was followed for

estimating protein in all the samples. Protein samples in the range of

(0-300 |xg) were taken in a set of tubes and final volume was made up to

1 ml with buffer. Five ml of alkaline copper reagent, prepared by mixing

0.5% (w/v) copper sulphate in 1.0% sodium potassium tartarate and 2%

(w/v) sodium carbonate in 0.1 N sodium hydroxide, in the ratio of 1:50,

was added to the tubes. This was followed by 10 min. incubation at room

temperature after which 0.5 ml of 1 N Folin's phenol reagent was added

with immediate mixing. The blue colour developed was read at 660 nm

after 30 min. against a blank prepared in similar manner except the

protein samples was replaced with buffer. The protein concentration of

various samples was calculated from the standard graph prepared using

30 mg/100 ml BSA as standard.

2.2.6 Electrophoresis

2.2.6.1 Non-denaturing Poly aery lamide Gel Electrophoresis

Non-denaturating PAGE was performed essentially according to

the method of Laemmli (1970) using slab gel apparatus manufactured by

Biotech India. A stock solution of 30% acrylamide containing 0.8%

bisacrylamide was mixed in appropriate order and proportion to give the

desired percentage of gel. It was then poured into the mould formed by

two glass plates (8.5 x 10 cm) separated by 1.5 mm thick spacers.

48

Bubbles and leaks were avoided. A comb providing a template for seven

wells was quickly inserted into the gel film and polymerization was

allowed to occur. After 15-20 minutes, the comb was removed and wells

cleaned and overloaded with running buffer. Samples containing 15-35

Jig protein mixed with equal volume of sample buffer (containing 10%

v/v glycerol, 0.0625 M Tris-HCl pH 6.8 and traces of bromophenol blue

as tracking dye) were applied to the wells. Electrophoresis was performed

at 100 V in electrophoretic buffer containing 0.025 M Tris and 0.2 M

glycine until the tracking dye reached the bottom of the gel.

2.2.6.2 SDS PAGE

SDS-PAGE was performed by the Tris-glycine system of Laemmli

(1970) using slab gel electrophoresis apparatus manufactured by Biotech,

India. 1.5 M Tris (pH 6.8 and 8.8) and 10% SDS were prepared and

mixed in appropriate proportion to give the final required percentage. It

was poured in the mould formed by two glass plates (8.5 x 10 cm)

separated by 1.5 mm thick spacers avoiding leaks and bubbles. A comb

providing a template for seven wells inserted into the gel before the

polymerization began, and removed once the polymerisation was

complete and the gel overloaded with the running buffer. Protein samples

were prepared in the sample buffer with 0.0625 M Tris-HCl, pH 6.8, 10%

v/v glycerol, 1% w/v SDS, traces of bromophenol blue as tracking dye

and where required 5% (v/v) (3-mercaptoethanol. The samples were kept

in boiling water bath for five minutes. Electrophoresis was performed in

the buffer containing 0.025 M Tris-0.2 M glycine at 100 V till the

tracking dye almost reached the bottom of the gel.

49

2.2.6.3 Staining procedure

After the electrophoresis was complete, the gels were removed and

protein bands visualized by staining with 0.1% commassie brilliant blue

R-250 in 40% (v/v) isopropanol and 10% (v/v) acetic acid. Destaining

was carried out by incubating the gels with 10% glacial acetic acid and

5% methanol at room temperature with shaking.

2.2.6.4 Agarose gel electrophoresis

Agarose gel electrophoresis of native or EcoRl/BamHl treated

DNA or plasmids were performed in a horizontal slab gel apparatus

manufactured by Biotech, India. Required amount of agarose was

suspended in IX TAE buffer (40 mM Tris, 0.9 mM EDTA, 24 mM acetic

acid pH 8.5) and dissolved by boiling the solution. The molten agarose

gel solution was then poured on to the casting plates prepared by

stretching adhesive tape on their sides to hold the molten agarose. A

teflon comb was fixed in place near one end of the plate, held about 1.0

mm above the plate and removed once the gel solidified in 15-20 min.

After stripping of the tape, gel was submerged in the electrophoresis tank

having IX TAE buffer. Appropriate amount of DNA, EcoRl/BamHl

digested linear DNA or plasmids and 1/10 volume of lOX tracking dye

(SDS 0,2%, sucrose 20%, Bpb 0.1%) added was mixed by tapping and

microfuged for 2-3 sec and loaded in the gel using a micropipette. The

samples were run into the gel for five min. at 50 V and then at a constant

current of 100 volts (Maniates, 1982).

50

2.2.6.5 Staining of DNA in agarose gel and visualization

In order to visualize the DNA bands in gels after electrophoresis

they were stained overnight with 1.0 \ig/m\ ethidium bromide and

photographed with type 55 Polaroid film under short wave UV

transilluminator (Suqden et al., 1975).

2.2.7 Assay of restriction endonucleases

For the assay of EcoRl and BamHl, endonuclease procedure of Lee

et al. (1978) was followed. The reaction mixture contained in a total

volume of 50 il, 10 mM Tris-HCl pH 8.0, 100 mM NaCl and 10 mM

MgCl2, 10 mM p-mercaptoethanol, 1 jxg of X DNA and 0.5 pig of plasmid

DNA and appropriate amount of the enzyme. The reaction mixture was

incubated for one hour at 37°C, stopped with dye reagent containing

0.12% SDS, 70% sucrose and 0.1% bromophenol blue. One unit is the

amount of enzyme needed to completely digest one microgram of DNA.

During the assay of immobilized restriction enzymes, the samples were

continuously agitated.

51

Hmmunoaffini Y immobilizafon of Ecom

RESULTS

3.1 Immunization of rabbits against EcoRl

A number of enzymes have been favourably immobilized on antibody

supports but to our knowledge work described in this thesis is the first

attempt of immobilization of restriction endonucleases with the help of

antibodies. EcoRl was immunogenic and rabbits challenged with the enzyme

responded by producing the good titre of antibodies against the enzyme. As

shown in Fig. 3.1, a single precipitin line was observed on immunodiffusion

of EcoRl against the antiserum.

3.2 ImmunoafGnity immobilization of EcoRl

The binding of the partially purified IgG fraction isolated from the anti

EcoRl antiserum on CNBr activated Sepharose 4B was performed by the

procedure described in previous sections and 20.7 mg/ml of the globulin

could be coupled to the matrix (Table 3.1). The matrix was highly effective

in the binding of EcoRl with nearly all the enzyme added bound leaving

very little in the supernatant and the washing. Of the 250 units of the enzyme

that was added to the support, more than 95% of enzyme was immobilized

The preparation obtained thus exhibited good restriction activity on X DNA

and the cleavage pattern indicates about 50 percent activity of the bound

preparation (Fig. 3.2). The figure also shows that the immobilized

preparation exhibited the characteristic cleavage of K DNA into fragments

that was indistinguishable from those of the soluble enzyme. It is well

52

Fig. 3.1 Immunodiffusion of anti EcoBl antiserum against EcoBI

Immunodiffusion was carried out in 1% agarose slabs as

described in the methods. The central well contained 280 units

of EcoRl while 28 [xl of the antiserum was placed in the

peripheral wells.

53

Table 3.1

Binding of partially purified IgG fraction on CNBr activated Sepharose

IgG added (mg)

IgG unbound (mg)

IgG immobilized (mg)

% Binding

44.0

23.3

20.7

45.9

each value represents the mean of at least two independent experiments performed in duplicate.

Fig. 3.2 Immobilization of EcoRI on the immunoaffinity support

Digestion of the X DNA was carried out using soluble or

immunoaffinity immobilized EcoRl as described in the text.

One fig of X DNA was incubated either with 4 units of soluble

EcoRl (Lane 2), or immobilized EcoRl (Lanes 3-5), 30 il

supernatant obtained after incubation of EcoRl with the

immunoaffinity support (Lane 6) or 30 \i\ of the first wash of

the enzyme after the immobilization process (Lane 7). Lane 1

contained "k DNA not exposed either to soluble or

immobilized EcoRl.

a [ Ace. No ^-. '

55

known that EcoRl cleaves X DNA to yield fragments of 21226, 7421, 5804,

5643, 4878 and 3530 bp with two oligonucleotides (5804 and 5643 bp)

exhibiting comparable mobility (Maniatis et ai, 1982).

3.2.1 Behaviour of Immobilized EcoRi

3.2.1.1 Restriction activity on plasmids

In further attempts to confirm lack of alteration in the specificity of

action of the antibody support bound £'coRI, its action on two different

supercoiled plasmids was investigated. These were pBR322, a 4.36 Kb

plasmid having a single cleavage site for EcoRl and a larger plasmid

pBHLUCP of 7.032 Kb also with a single site for EcoRl (kindly provided by

Dr. Arshad Rehman of Chicago University). Fig. 3.3 shows that action of

both the soluble and immobilized EcoRl was identical on the coiled and

supercoiled plasmids and both were characteristically linearized to a form

that exhibited migration between the slow open circular form and the fast

supercoiled form.

3.2.1.2 Stability of the immobilized EcoRl

3.2.1.2.1 Effect of exposure to various temperatures

Resistance of the immobilized EcoRl against inactivation at various

temperatures is shown in Fig. 3.4. Soluble EcoRl subjected to preincubation

at 37°C retained nearly full restriction activity on X DNA but was

readily inactivated on exposure to highertemperatures. As evident no DNA

cleavage was observed in lane 3-8 which contained X DNA treated with

56

Fig. 3.3 Digestion of the plasmid pBR322 and pBHLUCP by

soluble and antibody support immobilized EcoBl

Soluble or immobilized preparations of EcoRl (4 units) were

incubated with 0.5 \ig of the plasmids under standard

conditions and the digests subjected to electrophoresis. Lane

1- pBR322 alone, Lane 2- pBR322 + soluble EcoRl, Lane3-

pBR322 + immobilized EcoRl, Lane 4- pBHLUC P alone.

Lane 5- pBHLUC P + soluble EcoRl, Lane 6- pBHLUC P +

immobilized EcoRl.

57

Fig. 3.4 Effect of pretreatment at various temperatures on soluble

(A) and antibody support immobilized (B) preparations of

Ecom

In a total volume of 50 \il 4 units of soluble or immobilized

EcoRl were exposed to various temperatures for 30 min,

cooled for 60 min at 0°C and restriction activity determined on

X DNA under standard conditions. Lane 2- contained EcoRl

sample not subjected to the heat treatment, while those in

Lanes 3, 4, 5, 6, 7 and 8 contained samples exposed to 37°C,

45°C, 50°C, 55°C, 60°C and 65°C, respectively. Lane 1-

contained untreated k DNA.

58

1 2 3 A 5 6 7 8

B

1 2 3 A 5 6 7 8

EcoRl preincubated at 45°C, 50, 55, 60 and 65''C (Fig. 3.4, A). In contrast, a

nearly identical pattern of DNA cleavage was seen when X DNA was

incubated with immobilized enzyme exposed to temperatures up to 60°C for

30 min (Fig. 3.4, B). Interestingly some restriction activity was also

exhibited by the immobilized preparation incubated at 60°C or even 65°C.

3.2.1.2.2 Storage stability and reusability

Commercial EcoRl is quite labile at 4"C requires storage at -20''C.

The immobilized EcoRl was more stable to storage at the temperature. No

loss in activity of EcoRl immobilized on antibody support was detectable

over a period of 6 to 8 weeks of storage at 4°C as shown in Fig. 3.5. £'coRI

immobilized on antibody support was also reusable. The reusability was

investigated by separating the matrix bound enzyme from the assay mixture

by centrifugation prior to the assay, washing it thoroughly and resuspending

in fresh assay mixture. As shown in Fig. 3.6 there was no detectable loss in

activity after three consecutive assays.

3.2.2 Direct immobilization of EcoRL from the crude lysate of

E. coli pMB4

The binding of EcoRl by the antibody support was anticipated to be

highly specific and this property was used to immobilize the enzyme directly

from the crude lysate of an EcoRl over producing E. coli strain pMB4. The

strain has a combination of plasmids, which over produce EcoRl. Cell-free

lysate of the E. coli pMB4 was prepared as described in the text. The

59

Fig. 3.5 Effect of storage on the restriction activity of EcoRl

immobilized on the antibody Sepharose.

EcoRl immobilized on the anti EcoRl IgG support as

described in the text was suspended in 50mM Tris storage

buffer pH 7,5 at 4°C. The preparation was assayed after

having stored for 1 (Lane 4), 2 (Lane 5), 4 (Lane 6) and 6

(Lane 7) weeks. Lane 1 contained X, DNA alone and Lanes 2

and 3 contained A, DNA incubated with soluble and freshly

immobilized enzyme respectively.

60

Fig. 3.6 Reusability of the EcoBl immobilized on antibody support.

EcoRl immobilized as described was assayed under standard

conditions and separated from the reaction mixture by

microfuging for 1 min. The immobilized preparation was

washed 3 times with 50 mM Tris storage buffer pH 7.5 and

fresh assay mixture containing X DNA was added for the

subsequent assay. Lane 1 contained X DNA alone and Lane 2-

? DNA incubated with soluble £coRI Lane 3, 4, 5 and 6

composed of the X DNA samples treated with the immobilized

Bamm, for first, second, third and fourth time.

61

Sepharose support bearing 20.7 mg IgG/ml gel was incubated overnight with

the lysate at 4°C. After washing off the unbound and loosely bound

proteins, the support matrix was assayed for restriction activity on A. DNA.

The lysate exhibited characteristic EcoRl restriction pattern but resulted in

remarkable non-specific cleavage of DNA on incubation for longer

durations. This was apparently related to the presence of non-specific

nucleases in the lysate.

As shown in Fig. 3.7, the fragmentation pattern of the X DNA by the

immobilized preparation obtained by direct binding from the lysate was

identical with that prepared using pure EcoRl. The characteristic bands

resulting from the EcoRl action can be seen in the gel. More interestingly,

the non-specific digestion of k DNA that is striking after incubation with the

lysates with DNA was clearly absent from the sample incubated with the

immobilized enzyme.

62

Fig. 3.7 Time dependent cleavage of DNA by E. coli pMB4 lysates

(A) and the immobilized preparation (B)

Aliquots of E. coli pMB4 lysates or those containing the

immobilized preparation were incubated with X DNA for

various indicated intervals under standard conditions. Lane-1

contained A, DNA alone while DNA incubated with 4 units of

EcoRl was loaded in Lane-2. Lanes 3, 4, 5, 6, 7 contained the

X DNA incubated for 10,15, 30, 45 and 60 min, respectively

with the lysate (A) or the immobilized preparation (40^1) (B).

63

A

B

1 2 3 A 5 6 7

Hmmobilizotion oT Samlll on anHenzYme onHbody supports

4.1 Immunization of rabbits and isolation of IgG from the

antisera

Antibodies were raised against BamHl in rabbits as described under

methods. As in case of EcoRl, BamHl was also immunogenic although

additional booster doses were necessary to elicit the formation of good titre

of antibodies. On immunodiffusion of 5amHI against its antiserum, single

sharp precipitin lines were obtained (Fig. 4.1).

For the purification of anti 5amHI antibodies, the procedure described

by Catty and Raykundalia (1988) was followed. Globulin fraction of anti

BamHl antiserum was precipitated between 20-40% saturation of

ammonium sulphate, the precipitate dissolved in 20 mM sodium phosphate

buffer pH 7.2, and subjected to ion exchange chromatography DEAE

cellulose column (1.8 x 10 cm) equilibrated with same buffer as detailed in

methods.

Purification of IgG was followed with the help of polyacrylamide gel

electrophoresis performed both in the presence and absence of SDS. Whole

antiserum and the ammonium sulphate fractions showed bands

corresponding to IgG along with several others serum polypeptides (Fig 4.2,

lane 1, 2). All the additional polypeptides were removed after DEAE

cellulose chromatography and on electrophoresis the IgG purified thus gave

a single major band (Fig 4.2, lane 3).

To assess the purity of the IgG fraction, electrophoresis in the

presence of SDS was performed and the IgG migrated as two major bands

(Fig. 4.3, inset). In order to determine the molecular weight of the isolated

64

Fig. 4.1 Immunodiffusion of rabbit antiserum against BamHl

Antiserum raised against BamHl as described in methods was

used. Central well contained 300 units of BamHl and

peripheral wells 1, 2 and 3 contained 10, 20 and 50 [AI of

antiserum.

65

Fig. 4.2 Isolation of gamma globulin from sera of rabbit

immunized with the restriction enzyme BamHl

Lane 1- antisera (40 \xg). Lane 2- 40% ammonium sulphate

fraction (40 |ig). Lane 3- DEAL fraction (15 i g).

66

b

ny 1 2 3

Fig. 4.3 Molecular weight determination of IgG

The relative mobility of the standard marker proteins in the

SDS gel (inset) were plotted against logarithm of molecular

weight (Weber and Osbom, 1969) using least square analysis.

Arrows indicate the positions of the large and small molecular

weight peptides from the DEAE fraction.

Fraction from DEAE-cellulose and markers were heated with

the sample buffer at 100°C for 5 min and subjected to 12.5%

polyacrylamide gel electrophoresis in presence of SDS. Lane a

contained molecular weight markers (bovine serum albumin-

66.2 kDa, ova albumin-45 kDa, carbonic anhydrase-31 kDa,

trypsin inhibitor- 21.5 kDa, lysozyme-14.4 kDa, aprotenin-6.5

kDa). To the lane b was added 30 ^g DEAE-cellulose fraction.

67

0.4 0.6 0.8 1.0

Relative mobility

1.2 1.4

IgG, SDS- PAGE of sample was run along with marker proteins. The

molecular weight of IgG polypeptides was calculated by procedure of Weber

and Osbom (1969) with the help of a calibration curve in which the mobility

of markers proteins were plotted as a function of the logarithm of their

molecular weights (Fig. 4.3). The position of the migration of IgG subunits

corresponded to the apparent molecular weights of 51.28 kDa and 25.118

kDa for heavy and light chain respectively.

4.2 Immobilization of BamRl on Sepharose 4B matrix

precoupled with anti BamRI IgG

Purified anti BamHl IgG was coupled onto the CNBr activated

Sepharose as described earlier. The preparation obtained contained 19 mg

IgG bound per ml gel (Table 4.1). Immobilization of BamHl was achieved

by incubation of 250 units of the enzyme with 250 il of IgG-Sepharose

matrix. The enzyme bound support was isolated by centrifugation, washed

repeatedly with buffer and the matrix investigated for restriction activity on

K DNA. Figure 4.4 shows that while the supernatant contained barely

detectable restriction activity, the first wash did contain some suggesting the

presence of a small amount of loosely bound BamHl. The figure also shows

that the restriction activity of the preparation was comparable with that of

the soluble enzyme as apparent from the characteristic fragmentation of

X DNA. The X DNA has five restriction sites (5.5 Kb, 22.3 Kb, 27.9 Kb,

34.5 Kb and 41.7 Kb) of BamHl, some of which remained unresolved in the

gel. Lane 3, 4 and 5 contained respectively 5, 10 and 15 il of matrix and

show a concentration dependent increase in X DNA restriction.

68

Table 4.1

Binding of purified IgG fraction on CNBr activated Sepharose

IgG added (mg)

IgG unbound (mg)


% Binding

42.5

23.4

19.2

45.0


Fig. 4.4 Immobilization of BamHI on the anti BamHI IgG

Sepharose support.

Digestion of X DNA was carried out using soluble or

immunoaffinity immobilized BamHI as described in methods.

One [xg of X DNA was incubated with 5 units of soluble

BamHI (Lane 2) or 5 \i\ (Lane 3), 10 \x\ (Lane 4) and 15 |LI1

(Lane 5) of the immobilized preparation. Lane 6 contained the

X DNA incubated with 20 jil of supernatant obtained after

binding of BamHI to the support while the lane 7 contained 20

il of first wash incubated with the DNA.

*«==Ais

3 A 8

4.2.1 Behaviour of the immunoaffinity immobiHzed BamHl

4.2.1.1 Action on plasmids

The restriction activity of the immunoaffinity immobilized BamHl on

the plasmid pBR322 and pUC19 was also identical with the soluble enzyme

(Fig. 4.5). As evident the mobility of the supercoiled plasmid is fast and it

was transformed to the slow migrating form after restriction enzyme

digestion. The faint and very slow migrating bands appearing in the sample

not exposed to the BamHl may represent the open circular form resulting in

shear-induced nicks. These bands are however not evident in plasmid

preparations treated with either soluble or immobilized BamHl. Presumably

both super coiled and the open circular forms are transformed to the linear

form with mobility lower than the super coiled plasmid.

4.2.1.2. Effect of temperatures

BamHl immobilized on the antibody support was also clearly more

resistant to thermal inactivation than the native enzyme (Fig 4.6). The

soluble enzyme retained most activity after preincubation at 37°C, 45°C and

50°C but was inactivated when exposed to temperatures above 55°C for 30

minutes. The immobilized preparation showed very little loss in activity after

preincubation at temperatures up to 55''C and retained significant activity

even after incubation at 60°C. Both native and the immobilized preparations

were completely inactivated when exposed to a temperature of 65°C.

71

Fig. 4.5 Digestion of the plasmids pBR322 and pUC19 by soluble

and antibody support immobilized BamHl.

Soluble or immobilized preparations of BamHl (5U) were

incubated with 0.5 ig plasmids under standard conditions and

the digest was subjected to electrophoresis.

Lane 1- pBR322 alone, Lane 2- pBR322 + soluble BamHl,

Lane 3- pBR322 + immobilized BamHl, Lane 4- pUC19

alone, Lane 5- pUC19 + soluble BamHl, Lane 6- pUC19 +

immobilized BamHl.

72

Fig. 4.6 Effect of pretreatment at various temperatures of soluble

(A) and antibody support immobilized (B) preparations of

Bamm

In a total volume of 50 il, 5 units of soluble or immobilized

BamHl were exposed to indicated temperatures for 30 min,

cooled for Ih at 0°C and restriction activity determined on

k DNA under standard conditions. Lane 2 contained BamHl

sample not subjected to the heat treatment, while those in lane

3, 4, 5, 6, 7 and 8 contained samples exposed to 37°C, 45°C,

50°C, 60°C and 65°C respectively. Lane 1 contained

untreated k DNA

73

4.2.1.3 Storage stability and reusability

The long-term stability of the immobilized enzyme was investigated

by storing the preparation at 4°C. While storage up to 4 weeks resulted in no

inactivation, a fraction of activity appears to be lost in the preparations

stored for six and eight weeks (Fig, 4.7). The immobilized enzymes also

displayed no significant loss in activity when reused 3 times (Fig. 4.8).

4.3 Properties of BamHl immobilized on Sepharose matrices

bearing randomly coupled or favourably oriented antibodies

Considerable data has accumulated over the last few years that

advocate the advantage of favourably orienting antibodies on solid supports

for binding and immobilizing antigenic enzymes (Turkova, 1999). Attempts

were therefore made to orient anti BamHl IgG on Sepharose. For this

purpose, the procedure described by Hale (1995) in which the IgG are bound

on Co " loaded IDA Sepharose was used. The support has been shown to

bind IgG via Fc region only. Table 4.2 shows that Co^*-IDA Sepharose binds

only 3.4 mg IgG /ml. This was considerably lower than the amount that

could be immobilized on CNBr activated Sepharose. To be able to make a

meaningful comparison, Sepharose bearing same amounts of randomly

oriented IgG were prepared using the CNBr procedure. The two supports

were compared for the BamHl binding activity by separately incubating

them with 250 units of BamHl. The unbound enzyme was removed and the

matrices were thoroughly washed with buffer.

74

Fig. 4.7 Storage stability of immobilized BamUl at 4"C

The immobilized preparation of BamHl was stored at 4°C and

activity determined at different interval of time.

Lane 1 contained X DNA alone, Lane 2 that treated with

soluble BamHl. Lane 3 contained X DNA treated by freshly

immobilized BamHl. Lane 4, 5, 6 and 7 contained X DNA

samples treated with 20 \i\ of immobilized BamHl that were

respectively stored for 2, 4, 6 and 8 weeks.

75

1 2 3 ^ 5 6 7

Fig. 4.8 Reusability of the BamHl immobilized on antibody

support.

BamHl immobilized on the antibody support was incubated

with X DNA under standard conditions, and separated from

the reaction mixture, washed and suspended in fresh assay

mixture containing K DNA. The procedure was repeated for

three times. Lane Icontained untreated k DNA alone; Lane 2

contained A, DNA treated with soluble BamHl, Lane 3, 4 and 5

composed of the A, DNA samples treated with the immobilized

BamHl, for first, second and third time.

76

Table 4.2

2+ Binding of IgG on Co - IDA Sepharose

IgG added (mg)

IgG unbound (mg)


% Binding

9.0

5.6

3.4

38.0


> \"{ Ace. No )^ )

Both the randomly and favourably oriented IgG supports bound

BamHl and the degradation of the X DNA by the immobilized preparations

increased with the amount of matrix used (Fig. 4.9).

That the favourably oriented antibody support exhibited enhanced

binding of BamHl is evident from the Figure 4.10. Lanes 3-8 contained

X DNA exposed to increasing amounts of the immobilized preparation. No

detectable activity was evident when up to 20 [0,1 of the randomly oriented

preparation treated k DNA and the restriction activity was barely detectable

in the supernatant that contained k DNA exposed to 25 il of the matrix.

Further increase however resulted in marked increased in the restriction

activity and nearly all the DNA disappeared from the samples containing

30 \x\ of the matrix. In contrast small amount of restriction activity could be

observed even in the 5 \i\ sample bearing BamHl favourably immobilized on

oriented antibodies. Nearly all DNA was degraded in the sample incubated

with 20 \i\ of the immobilized preparation.

4.3.1Action on plasmids

The action of ^arnHl immobilized on randomly coupled or favourably

oriented antibody support is shown in Fig. 4.11, A and B). As observed

earlier both the immobilized preparations were indistinguishable with

regards to their action on the two plasmids and transformed them to linear

forms.

78

Fig. 4.9 Immobilization of BamHI on supports bearing comparable

amounts of anti BamHI IgG linked to cyanogen bromide

activated Sepharose (A) or bound on cobalt charged IDA-

Sepharaose (B)


immunoaffinity immobilized BamHI as described in method.

One |LXg of X, DNA was incubated with either 5 units of soluble

BamHI (Lane 2) 25JA1, 30JX1 and 40^1 of immobilized BamHI

(Lane 3, 4, 5). Twenty pil of supernatant obtained after incubation

of BamHI with the immunoaffinity support (Lane 7) and second

wash of the enzyme (Lane 8) Lane 1 contained k DNA not

exposed to either soluble or immobilized BamHI.

79

1 2 3 4 5 6 7 8

B

1 2 3 4 5 6 7 8

Fig. 4.10 Concentration dependent cleavage of X DNA by

immobilized BamHl preparation

BamHl was immobilized on Sepharose supports bearing anti

Bamm IgG coupled by CNBr activation (A) or Co^^-IDA (B).

X DNA was incubated with various concentration of the

immobilized preparation and subjected to electrophoresis.

Lane 1 contained X DNA alone. Lane 2 contained X DNA

incubated with soluble BamHl, Lane 3, 4, 5, 6, 7and 8

contained X DNA treated with 5 ^1, 10 AI, 15 yd, 20 il, 25 (xl

and 30 \i\ of immobilized preparations respectively.

80

1 2 3 A 5 6 7 8

B

1 2 3 A 5 6 7 8

Fig. 4.11 Digestion of the plasmids by BamHl linked to IgG

immobilized on CNBr activated (A) and metal chelated

support (B)

Soluble or two different immobilized preparations having

equal amounts of IgG (3.4 mg IgG/ml) linked to BamHl were

incubated with 0.5 ig of plasmids under standard conditions

of the digest, subjected to electrophoresis. Lane 1- pBR322

alone, Lane 2- pBR322 + soluble BamHl; Lane 3- pBR322 +

immobilized preparation of BamHl; Lane 4- pUC19 alone,

Lane 5- pUC19 + soluble EcoRl; Lane 6- pUC19 immobilized

BamHl.

81

1 2 3 A 5 6

B

1 2 3 4 5 6

4.3.2 Effect of temperatures

A comparison of resistance to inactivation of BamHl preparation

immobilized on randomly coupled or favourably oriented support indicated

that the former is more stable against thermal inactivation (Fig. 4.12, A). The

favourably oriented support bound BamHl was nearly completely

inactivated even at 55°C as evident from the Fig. 4.12, B.

82

Fig. 4.12 Effect of pretreatment at various temperatures of BamHl

immobilized on IgG-Sepharose (A) or IgG CO^*-IDA

Sepharose (B)

In a total volume of 50^il 5 units of BamHl was exposed to

indicated temperatures for 30 min, cooled for 1 h at 0°C and

activity determined by using X DNA as substrate under

standard conditions. Lane 2 contained BamHl not subjected

to preincubation Lane 3, 4, 5, 6, 7 and 8 contained samples

exposed to 37°C, 50°C, 55°C, 60°C and 65°C respectively.

Lane 1 contained A, DNA alone.

83

^««W'ywlgW

1 2 3 A 5 6 7 8

B

1 2 3 4 5 6 7 8

K!!initY imrnQbilization o! a geneHcolly engineered Ijybrid prol'ein

5.1 The recombinant organism E. coli JM109 expressing the

fusion protein SpA::EcoRI

A recombinant strain E. coli JM109 (3P) harbouring a combination of

repressor plasmid pRK248cI, the protection plasmid EcoR4 and the

production plasmid pMTC48 was kindly provided by Prof. K. Schuegerl and

J. I. Rhee of the Institute of Technical Chemistry University of Hannover,

Germany. The organism synthesizes the fusion protein SpA::EcoRl the

product of fusion of staphylococcus protein A (a receptor for the Fc domain

of immunoglobulin G) and the restriction endonuclease ^coRI. In view of

the strong affinity between the protein A from the recombinant E. coli

JM109 and Fc fragment of IgG (Nilsson et al., 1985; Maschke et ai, 1992),

a solid support with covalently bound IgG was prepared for the affinity

immobilization of SpAiEcoRl

5.2 Preparation of immobilized goat IgG

The purified IgG was immobilized on cyanogen bromide activated

Sepharose as described earlier to give 18 mg IgG per ml gel (Table 5.1).

5.3 Fractionation ofE. coli JM109 lysate

The E. coli JM109 was grown overnight at 30°C and the cultures

were further incubated at 38°C for 4 h for the optimum induction of the

fusion protein SpAiEcoRl (Brandes et al., 1993). The cells were then

harvested by centrifugation at 9800 x g for 30 min. The pellet obtained was

suspended in a minimum volume of TA buffer and then in the lysis buffer as

84

Table 5.1

Binding of purified IgG on HSA* Sepharose

IgG fraction added (mg)

IgG unbound (mg)


% Binding

24.3

11.2

13.1

53.9

* 18 mg of HSA bound/ml of gel.


described in methods to achieve cell lysis. The lysate was then centrifuged at

9800 X g for 30 min. to remove cell debris. The EcoRl activity was

determined in the lyaste using X DNA substrate. As evident from the Fig. 5.1

that EcoRl activity was detectable in lysates but due to the presence of non

specific nucleases, the characteristic restriction pattern on X, DNA was not

detectable, especially in samples bearing higher concentration of lysates

(Fig. 5.1). The cell free lysate was therefore first subjected to fractionation

using streptomycin sulphate followed by ammonium sulphate fractionations.

As seen from the Figure 5.2, the restriction activity was not evident to any

significant extent in 0-25% precipitate, while nearly all of it was precipitated

when ammonium sulphate saturation was raised to 50%. The 25-50%

saturated ammonium sulphate fraction was therefore used as the source of

enzyme for the immobilization on the IgG support.

5.4 Affinity immobilization of SpA:: EcoBl

The dialysed 25-50% ammonium sulphate fraction from E. coli

JM109 was mixed with 0.5 ml IgG-Sepharose 4B. After 16 h of incubation,

the solid particles were collected and washed repeatedly with 10 mM

phosphate buffer pH 7.2, 0.5 mM EDTA and 50% glycerol, to remove the

adhering proteins from the IgG support matrix. The preparation obtained

thus exhibited high EcoRl activity as shown in Figure 5.3. The restriction

activity expressed by the immobilized enzyme was characteristically that of

EcoRl (Fig. 5.3, lane 1).

86

Fig. 5.1 EcoRI activity in the lysates ofE. coli JM109

Aliquots ofE. coli JM109 (3P) lysates were incubated with X

DNA under standard conditions subjected electrophoresis on

1.0% agarose gel. Lane 1- X DNA alone; Lane 2- X DNA

incubated with 4 units of soluble EcoRl; Lane 3- 7 contained

the X DNA incubated respectively with 20, 25, 30, 35 and

40jil of lysate.

Fig. 5.2 Ammonium sulphate fractionation of the lysate of E. coli

JM109

The lysate of E. coli JM109 prepared as described in methods

was subjected to 0 - 25% and 25 - 50% ammonium sulphate

fractionation. The dialysed precipitates and supematants were

assayed for £'coRI activity under standard conditions. Lane

1- X DNA alone, Lane 2- X DNA digest of soluble EcoRl, Lane

3- precipitate of 0 - 25% ammonium sulphate fraction; Lane

4- supernatant of the 0 - 25% ammonium sulphate fraction;

Lane 5-precipitate of 25 - 50% ammonium sulphate cut, Lane

6- supernatant of the 25 - 50% ammonium sulphate fraction.

88

Fig 5.3 Immobilization of fusion protein £coRI::SpA on

randomly coupled IgG support


randomly immobilized £coRI::SpA as described in methods.

A, DNA was incubated either with 4 units of soluble EcoRl

(Lane 2) or 30 \x\ of the immobilized EcoRl (Lane 3, 4, 5),

Lane 1 contained X DNA not exposed either to soluble or

immobilized EcoRl

89

1 2 3 A 5

5.4.1. Activity on the plasmids

Super coiled plasmids pBR322 and pUC19 were nicked equally well

by the immobilized preparations and the activity was indistinguishable from

that of the soluble EcoRl as evident from Fig. 5.4.

5.4.2 Stability of immobilized preparation

The resistance of EcoRl activity of the immobilized fusion protein

against thermal denaturation was also studied (Fig. 5.5), the support bound

enzyme acquired greater stability and was inactivated at temperatures above

50°C. Non-specific cleavage of X DNA occurred at higher temperatures (Fig.

5.5, lanes 5, 6, 7 and 8). Soluble EcoRl is readily inactivated to

temperatures above 40°C (Fig. 3.4).

5.5 Oriented immobilization of the fusion protein

In an attempt to favourably orient the fusion protein on solid support

the IgG isolated from the sera of rabbit immunized with HSA were bound to

Sepharose support precoupled with HSA. This was expected to result in

good accessibility of Fc portion of the bound IgG for the binding of the

fusion protein. Excess of dialysed 25-50% ammonium sulphate fraction of

SpA::£'coRI lysate was incubated with the matrix at 4°C overnight and the

matrix was collected and washed with excess of potassium phosphate buffer

in order to remove the adhering proteins. The matrix was then washed with

Tris buffer pH 7.5 and then equilibrated with 25 mM Tris buffer containing

20% glycerol.

90

Fig. 5.4 Digestion of plasmids pBR322 and pUC19 by soluble and

antibody support immobilized fusion protein

Soluble or immobilized preparations of EcoRl (4 units) were

incubated with 0.5 [ig of the plasmids under standard condition

and the digest subjected to electrophoresis. Lane 1- pBR322

alone; Lane 2- pBR322 + commercial EcoRl; Lane 3- pBR322

+ immobilized EcoRI; Lane 4- pUC19 alone; Lane 5- pUC19

+ commercial ^coRI; Lane 6- pUC19 + immobilized EcoRl.

91

Fig 5.5 Effect of pretreatment at various temperatures on activity

of immobilized Eco RI

In a total volume of 50 yd, 40 (xl of immobilized preparations

were exposed to various temperatures for 30 min, cooled for

60 min at Q°C and restriction activity determined on k DNA

under standard conditions. Lane 2 contained commercial

EcoRl sample not subjected to the heat treatment, while those

in Lanes 3, 4, 5, 6, 7, and 8 contained samples exposed to

37°C, 45°C, 50°C, 55°C, 60°C and 65°C, respectively. Lane 1

contained X DNA alone.

92

1 2 3 A 5 6 7 8

The activity of EcoRl immobilized on the support was determined on

agarose gel using X DNA as substrate (Fig. 5.6). As evident from the Figure

the immobilized fusion protein preparation showed non-specific cleavage

pattern that suggested inactivation of the EcoRl activity. Activity on the

plasmids pBR322 and pUC19 however was characteristically

indistinguishable from that of the soluble enzyme (Fig 5.7).

93

Fig. 5.6 Immobilization of EcoRI::SpA on anti HSA IgG

favourably oriented on HSA Sepharose.

One [xg of X DNA incubated with the 4 units of soluble

and immobilized preparation under standard

conditions as described earlier. Lane 1 contained

X DNA alone, Lane 2- X, DNA incubated with soluble

EcoRl, Lane 3, 4, 5, 6, 7, X DNA treated with

orientedly immobilized EcoRl.

94

li»BiiP^

1 2 3 A 5 6 7

S6

pazTjTqouiuiT + 6X30^ -9 9U^1 'IÔD^ yBpiomuioo + 6X30^ -g

•1^03^ iBiDJouiuioo + zênad -z 9uî '9U0IB 33eâd -x ûî

;joddns QSJ pa;n9uo iCiqejnoAej no

pdzinqouimi v^S-'IH^^^^ P"" aiqnios qjiAi spiuiseid jo 38BAB3I3 /,*g 'Sij

Discussion

Discussion

Class II REs have several important applications- are highly labile

and continue to remain expensive -reasons sufficient to attempt their

immobilization. Indeed some studies describing the immobilization of REs

by covalent coupling (Lee et al, 1978), adsorptions to solid supports

(Cashion et al, 1982a) and by crosslinking with bifunctional reagents

(Dubey et al, 1989) are available. While the studies have claimed varying

degrees of success, the principal limitations appear to be the resulting

inactivation due to the modification of side chains of amino acid residues

and/or limited access of the immobilized enzyme to large molecular weight

DNA substrates.

Immobilization of enzymes on antibody supports does not involve

chemical modification of enzyme and the antibodies. In view of their large

dimensions they behave as large spacers between the solid support and the

enzyme (Silman and katchalski, 1986; Ehle and Horn, 1990) remarkably

improving the access of the immobilized enzyme (Shami et al., 1989).

Taking this and the fact that binding of the enzyme to appropriate antibodies

have been shown to remarkably improve their resistance to several forms of

inactivation (Saleemuddin, 1999; Turkova, 1999), an attempt was made to

investigate the potential of polyclonal antibodies in the effective

immobilization of the REs - £coRI and BamWl. An attempt has also been

made to immobilize SpA::£'coRI -a fusion protein produced by a

recombinant organism E. coli pMB4.

The EcoRl was immunogenic and induced the formation of

antibodies in rabbits readily in 4 weeks. As observed by several others

96

(Cinader, 1967; Pollock et al, 1967; Ben-Yousef e a/., 1975; deAlwis et ai,

1987) the antibodies were non-inhibitory. Some plausible explanations that

can be offered for the absence of inhibitory antibodies in the antisera include

the active site acting as blind spot for the immune system and steric

hindrance due to binding of antibodies recognizing adjacent locations of the

active site of the enzyme (Shami et al., 1989).

Enzymes bound to antibody supports generally exhibit high activity,

due to the antibody molecules acting as large spacer between the support and

the enzyme (Solomon et al., 1986; Shami et al., 1989; Shami et al., 1991).

The observed relatively lower (50%) activity of the antibody support bound

EcoRl activity despite the non-inhibitory nature of the antibodies, may be

related to steric hindrance on the diffusion of the very large molecular

weight of DNA used as substrate. It is likely that some of the immobilized

antibody molecules may bind EcoRl molecules in orientations that restrict

their access to the large molecular weight DNA. Other molecules of the

enzyme may be more accessible to substrate, as evident from the

fragmentation pattern of the DNA which was identical to that obtained with

the soluble EcoRl (Fig. 3.2).

The specificity of EcoRl immobilized on the antibody support

remained unaltered and the preparation exhibited characteristic restriction

activity not only on X DNA but also on the supercoiled and open circular

(occur due to shearing forces) plasmids pBR322 and pBHLUC bearing

single restriction site, which were linearized (Fig. 3.3). This suggested the

access of this enzyme towards both linear and coiled DNAs.

Thermal stability of EcoRl immobilized on antibody support was

remarkably enhanced and the enzyme retained nearly complete activity even

97

after 30 min. preincubation at 55°C. Soluble enzyme on the other hand lost

most restriction activity on exposure to temperature over 40°C (Fig. 3.4). It

is interesting to note that the immobilized preparations incubated at 60°C or

even at 65°C also show small but significant restriction activity and no star

activity (Thomas and Davis, 1975). Because the EcoRl preparations were

exposed to various temperatures and cooled prior to assay under standard

conditions, the observed stability may also be related, at least in part to the

ability of the immobilized enzyme to regain activity after thermal

denaturation (Horowitz and Bowman, 1987). In case of some enzymes

immobilization has been shown to facilitate refolding after denaturation by

restricting protein-protein interaction (Sinha and Light, 1975).

Both polyclonal (Sato and Walton, 1983; Jafri et al., 1993;

Siemann et al., 1994 Jafri et al., 1995) and monoclonal antibody (Turkova,

1999) supports have been shown to be effective in enhancing the resistance

to denaturation of a number of enzymes. Antibody binding appears to

involve reasonably large areas of protein antigen (Davis et al, 1988), which

may comprise of more than one polypeptide chain, and the stability

enhancement may consequently arise out of a crosslinking like effect of

antibody binding (Hansen and Beavo, 1982; Emst-Cabera and Wilchek,

1988). It has been hypothesized that denaturation may begin at a specific

area on the enzyme. If some of the antibodies are directed against such

regions they may confer remarkable resistance against unfolding of the

protein (Jones and Thronton, 1986; Sheriff et al, 1987; Ulbrich-Hoffman et

al., 1993; Arnold et al, 1996). Additional stabilization may also result due

to the enhancement in the rigidity of the enzyme conformation resulting

from the binding of a single enzyme molecule to more than one immobilized

98

antibody molecule (Sadana and Madgula, 1993). The antibody support-

associated EcoRl was also stable to storage for several weeks as evident

from Fig. 3.5 and could be reused several times (Fig. 3.6).

In view of high degree of specificity of interaction between

antibody and antigen, the feasibility of using the immunoaffinity adsorbent

for directly immobilizing £'coRI from a cell lysate was investigated. For this

purpose, the antibody support was incubated with the cell lysate of E. coli

pMB4, a strain that overproduces EcoRl. The matrix bound EcoRl was

washed and when incubated with X DNA, a fragmentation pattern identical

with that produced by soluble EcoKl resulted. More interestingly, the non

specific digestion of the "k DNA that is strikingly evident after incubation of

pMB4 lysate with the X DNA was clearly absent in the samples incubated

with the immobilized enzyme (Fig. 3.7). This indicates specificity in the

binding of EcoKl on the antibody support. The possibility of non-specific

association of some lysate proteins not related to DNA cleavage with the

support during incubation with the lysate cannot be ruled out also due to the

polyclonal nature of the antibody preparation used. It should however be

possible to minimize or eliminate such possible association using highly

purified/monoclonal antibodies. Direct immobilization of EcoKl from the

cell lysate offer the remarkable possibility of cutting down the cost of the

preparation of the immobilized enzyme since pure enzyme is not required.

Other instances of the use of polyclonal/monoclonal antibody support in the

direct immobilization enzymes from cell lysates are also available (Nilsson

et ai, 1985; Baneyx et al., 1990).

While it may be possible to improve the performance of

immobilized enzyme by replacing the polyclonal antibodies with appropriate

99

monoclonal antibody, the former if non-inhibitory as in the present study

may offer the advantage of lower cost and high stability. Monoclonal

antibodies have also been shown to be more labile than the polyclonal

antibodies (deAlwis and Wilson, 1989). Interesting strategies for raising

non-inhibitory polyclonal anti-enzyme antibodies in animals are also

available (Fusek et al., 1988; Shami et ai, 1991; Stovickova et al., 1991;

Jafri et al., 1993).

Studies conducted on the immobilization of BamVil on the anti

BamHl antibodies also yielded comparable results and indicated usefulness

of polyclonal antibody supports. The antiserum was also non-inhibitory

towards BamHl and the immobilized preparation of the enzyme active and

exhibited improved stability.

The anti BamHl antibodies raised in rabbits were non-inhibitory and

of precipitating type (Fig. 4.1). In order to eliminate non-specific proteins

from the support the ammonium sulphate fraction was further subjected to

purification on DEAE cellulose to obtain pure IgG fraction (Fig. 4.2 and Fig.

4.3). The IgG was immobilized on Sepharose supports. The fragmentation

pattern of K DNA by soluble BamHl and that bound on the antibody support

was indistinguishable and restriction activity showed a concentration

dependent increase over the range investigated (Fig. 4.4). The antibody

support bearing 19 mg/gm of IgG bound (Table 4.1) 200 units of 5amHI and

most of the activity appears to be expressed. The restriction activity of

BamHl immobilized on antibody support was also indistinguishable from the

soluble enzymes when coiled/super-coiled plasmids were used as substrates.

Both pBR322 and pUC19 that contain a single restriction site for BamHl

(Sambrook et al., 1989) were readily linearized on incubation with the

100

immobilized BamUl (Fig. 4.5). The good accessibilities of the enzyme and

the lack of alteration in the action are evidently related to the antibody

molecules acting as large spacers (Solomon et ai, 1986).

A comparison of resistance to thermal inactivation of soluble and

the antibody support immobilized BamHl is shown in Fig. 4.6. Native

BamHl was reasonably stable to a 30 min. exposure to temperature up to

60°C but lost most restriction activity when the incubation temperature was

increased to 65''C. The immobilized BamHl however remained active after

incubation at 65°C as well. Incubation at higher temperatures however

resulted in the inactivation of the enzyme. Thus as in case of EcoRl,

polyclonal antibodies raised in rabbits appear to confer remarkable stability

to the BamHl. In addition, the immobilized preparation could be stored at

4''C for about 2 months (Fig. 4.7) and reused for at least 3 times (Fig. 4.8).

A number of investigations have demonstrated the effectiveness

of favourably oriented antibodies in the binding of antigenic enzymes

(Subramanian and Velande, 1992; Bilkova et ah, 1999). Favourable

orientation of antibodies on solid supports may be particularly useful for

enzymes acting on large substrates, because of the greater problem of steric

hindrance. For example, antibodies immobilized by the Fc portions have

been shown to have twice the capacity of binding to antigens as compared to

the randomly immobilized antibodies (O'Shannessy and Wilchek, 1990).

Favourable orientation of the antibodies on the solid supports has

been shown to enhance the binding capacities of a variety of

immunoadsorbents (Hoffman and O'Shannessy, 1988; Prisyaznoi et al.,

1988; Turkova, 1993). This has been achieved by connecting the IgG to the

supports via their Fc regions with the help of protein A, (Solomon et al.,

101

1984) or linking via -SH group (Prisyaznoi et al, 1988) or carbohydrates

(Hoffman and O'Shannessy, 1988; Prisyaznoi et al, 1988) located around

the Fc region. More recently Hale and Beidler (1994) have shown that IgG

bind through their Fc regions to cobalt loaded IDA-supports and the

association rendered very strong and nearly irreversible by oxidizing the

bound Co^* to Co^* subsequent to the binding of the antibody. The strategy

was employed for favourably orienting the anti BamY{\ IgG. The procedure

however resulted in binding of far lower amounts of IgG than that

immobilized with the CNBr procedure (Table 4.2).

In order to compare the behaviour of the enzyme bound to

Sepharose supports bearing randomly linked or favourably oriented

antibodies, matrices bearing comparable amounts of immobilized IgG were

used. Since cobalt supports bound only 3.4 mg IgG/ml gel, equal amount of

IgG was added for immobilization on Sepharose support treated with excess

of CNBr. This resulted in quantitative coupling of added IgG to the support.

Figure 4.9 and 4.10 shows that BamYil immobilized on support bearing

favourably oriented IgG shows clearly greater restriction activity on X. DNA

as compared to that bound to the support bearing randomly oriented

antibody. Thus coupling of BamYil to IgG-Co^*-IDA Sepharose resulted in a

better efficiency of binding and higher activity than coupling that exhibited

on those CNBr Sepharose (Fig. 4.10). Also Bamm bound to Co^^-IDA

Sepharose was more active than that prepared by using IgG on CNBr

Sepharose. BarnRl immobilized on oriented antibodies exhibited good

restriction activity when tested on the plasmids pBR322 and pUC19 (Fig.

4.11). These results substantiate the earlier observations that antibodies

immobilized via Fc region may bind higher amounts of antigenic enzyme

102

(Fleminger et al., 1990). Thermostability of the BamUl immobilized on the

support bearing oriented antibodies was however surprisingly lower (Fig.

4.12). The reason for the lability of immobilized BamEl on Co^^-IDA

Sepharose may not be related to the low concentration of antibodies on the

support since the BamHl preparation immobilized on support bearing

comparable amount of antibodies linked via CNBr was more stable.

Lowered interaction with the support matrix and or limited association of the

oriented antibodies resulting in decreased lateral interactions of bound

enzyme with other immobilized antibodies are some of the plausible

explanations which however need substantialization. It is also likely that the

metal ions bound to support may be released during exposure to high

temperatures and cause enzyme inactivation (Ljunquist et al., 1989; Carlsson

etal., 1996).

A number of examples of manipulation of cloned genes for

facilitating protein purification are presently available. These are generally

accomplished by adding DNA encoding an additional oligopeptide/

polypeptide at the 5' or 3' end of the gene. Sometimes a gene encoding for

entire domain or even the complete protein is fused with gene of interest and

expression of the fused gene results in a fusion protein comprising of the

desired protein and a purification tag. The protein tag facilitates usually

single step purification by ion exchange; hydrophobic, covalent or metal

chelate separations (for review see Sassenfeld, 1990). The addition of

specifically designed tags or the modification of sequences within the target

gene product has enabled the development of novel strategies for

downstream processing that can be employed for efficient recovery of both

native or modified proteins.

103

More recently affinity tags of fusion protein have also been

exploited for the direct immobilization of fusion protein for achieving

improved transformation of the substrate of the enzyme component. These

include the immobilization of cellulose binding domain (CBD)-|3

glucosidase (Ong et ai, 1991) and CBD-heparinase (Shipigel et al., 1999)

on cellulose supports, several enzymes bearing polyhistidine tags (Horiuchi

et ai, 1988) and protein A- p galactosidase (Yang et ai, 1995) and protein

A- (3 Lactamase (Banyx et ai, 1990).

A fusion protein SpAi^EcoRI that exhibited characteristic EcoRl

restriction activity without the removal of Protein A is synthesized by E. coli

JM109 (Rhee et al, 1994). The strain harbours a combination of repressor

plasmid pRK248CI, the protection plasmid EcoRA and the production

plasmid pMTC48 (Maschke et al, 1992). The lysate oiE. coli JM109 show

EcoRI activity but was masked by the presence of some non-specific

nucleases (Fig. 5.1). Attempts were made to immobilize the fusion protein

from the 25-50% saturated ammonium sulphate fraction of the lysate of E.

coli JM109 (Fig.5.2). As shown the fusion protein could be successfully

immobilized on IgG-Sepharose to yield an active EcoRl preparation that

exhibit restriction activity comparable to the soluble enzyme on the linear

X, DNA (Fig. 5.3) and the supercoiled plasmids (Fig. 5.4). The resistance to

thermal inactivation of the ^coRI activity of the immobilized preparation

was enhanced (Fig. 5.5) although the enhancement appear to be of lower

magnitude as compared to that of ^coRI immobilized on anti EcoRA

antibody support (Fig. 3.2). Direct interaction of the immobilized antibody

with EcoKl in case of the later may be cause of higher stability. Attempts to

improve the performance of the immobilized SpA::£'coRI on oriented IgG

104

support were unsuccessful and the enzyme appeared inactive as evident by

its non-specific cleavage of the X DNA (Fig. 5.6) but with plasmids no

alteration in the action was found (Fig. 5.7). No plausible explanation for the

observation can be given especially considering that the enzyme

immobilized on the IgG-Sepharose was active and stable.

In conclusion it has been shown that polyclonal antibody supports

can be used for the effective immobilization of EcoRl and BamlU and the

preparation used for restriction of large and small molecular weight DNAs.

The preparations also exhibited thermal and storage stability. Applicability

of the procedure to other REs however requires specific investigations.

Considering the differences in the structure and action of EcoRl and Bamlil

and usefulness of the polyclonal antibody support wider applicability of the

strategies for other REs is not unlikely.

105.

Summary

Summary

Polyclonal antibody based procedures were developed for the

immobilization of the restriction enzymes EcoRl and BamHl and the

immobilized preparations were investigated for their activity, stability and

reusability.

Polyclonal antibodies were raised against EcoRl in rabbits and the

gammaglobulin fractions isolated from the immune sera. The gammaglobulins

were immobilized on CNBr activated Sepharose 4B. The matrices prepared

thus were effective in the binding of £'coRI. The antibody support immobilized

EcoRl exhibited restriction activity on the linear X DNA and plasmids

pBR322 and pBHLUC that was indistinguishable from that of the native

enzyme. The EcoRl bound to the antibody support also exhibited superior

thermal stability and retained specific restriction activity even after exposure to

55°C. It was also possible to directly immobilize EcoRl from the crude lysates

of E. coli pMB4, an EcoRl overproducing strain, to obtain an active

immobilized preparation.

Comparable behaviour was also exhibited by BamYll immobilized on the

antibody support. An immunoaffinity support for the immobilization of BamRl

was prepared by coupling the purified IgG fraction isolated from the anti

BamYll antiserum raised in rabbits. As in case of EcoRl, BamYll immobilized

on the antibody support exhibited high restriction activity on X DNA and

linearized the plasmids pBR322 and pUC19. The immobilized BamYll

preparation was remarkably more resistant to thermal inactivation as compared

to the native enzymes.

The usefulness of preparing a support with favourably oriented

antibodies for the immobilization of BamYll was also investigated. For this

106

purpose the anti BamUl antibodies were immobilized on the Co^^-IDA

Sepharose and their association with support strengthened by treatment with

H2O2 that oxidizes the matrix bound Co^* to Co^ . The procedure however

resulted in the binding of far lower amount of IgG. For comparison same

amount of IgG were coupled randomly to Sepharose using the CNBr

procedure. The two matrices were compared for their ability to bind BamHl. It

was observed that the support with favourably oriented IgG bound significantly

higher amount of enzyme as evident from its action on linear X DNA and the

super coiled plasmids pBR322 and pUC19. The BamHl immobilized on the

favourably oriented antibodies was however more labile against thermal

inactivation than the preparation immobilized on support bearing randomly

immobilized IgG.

The fusion protein SpA::£^coRI synthesized by the recombinant strain

E. coli JM109(3P) was also immobilized on IgG supports. The supports were

prepared either by coupling the goat IgG to CNBr activated Sepharose or

binding of rabbit anti HSA IgG on to Sepharose precoupled with HSA. The

later procedure was expected to result in orientation of IgG in manner that

facilitated good accessibility of the Fc region. The fusion protein could be

immobilized on the goat IgG support by incubating the latter with 25-50%

ammonium sulphate fraction to yield a preparation with characteristic EcoK\

restriction activity on linear X DNA and the plasmids pBR322 and pUC19.

Compared to the native enzyme the immobilized preparation also exhibited

improved resistance to thermal inactivation. The EcoKl bound to the

favourably oriented rabbit IgG inexplicably appeared to loose its specificity

when tested on X DNA although it linearized the plasmids pBR322 and pUC19

in the characteristic manner.

107

Sibliography

Aggarwal, A.K. (1995) Curr. Opin. Struct. Biol., 5, 11.

Agnellini, D., Pace, M., Cenquanta, S., Gardana, C , Pietta, P.G. and Mauri, P.L.

(1992) Biocatalysis, 6, 251.

Amarant, T. and Bohak, Z. (1981) Appl. Biochem. BiotechnoL, 6, 237.

Anderson, J.E. (1993) Curr. Opin. Struct. Biol., 3, 24.

Anspach, F.B. and Altmann-Haase, G. (1994) Boitechnol. Appl. Biochem., 20,

323.

Arnold, F.H. (1991) Biotechnology, 9,151.

Arnold, U., Rucknagel, K.P., Schierhorn, A. and Ulbrich-Hoffman, R. (1996)

Eur. J. Biochem., 237, 862.

Arnon, H. (1973) Immunochemistry of Enzymes, In: The Antigens (Sela, M. ed.).

Academic press. New York, 1, 88.

Askari, A., Hwang, W.H. and Antieu, J.M. (1980) Biochemistry, 19, 1132.

Axen, R. and Ernback, S. (1971) Eur. J. Biochem., 18, 351.

Baneyx, P., Schmidt, C. and Georgine, G. (1990) Enzym. Microb. Technol., 12,

337.

Bennett, S.P. and Halford, S.E. (1989) Curr. Top. Cell. Req., 30, 57.

Ben-Yousef, Y., Geiger, B. and Arnon, R. (1975) Immunochemistry, 12, 221.

Bernard, H.U., Remaut, E., Vickers, H.M., Das, H.K. and Helinsky, D.R. (1979)

Gene, 5, 59.

Bickerstaff, G.F. (1980) Int. J. Biochem., 11, 201.

Bickerstaff, G.F. (1982) In: Enzyme and Fermentaton BiotechnoL (Wiseman, A.

ed.) 9, 162.

Bickle, T.A., Pirrota, V. and Imber, R. (1980) Methods in Enzymol., 65,132.

108

Bilkova, Z., Mazurrouva, J., Churacek, J., Horak, D. and Turkova J. (1999) J.

Chromatogr., 852,141.

Bingham, A.H.A., Atkinsan, T., Sciddy, D. and Roberts, R.J. (1978) Nucleic

Acid Res., 5, 3457.

Bircakova, M., Tniksa, M. and Scoulen, W.H. (1996) J. Mol. Recognit., 9, 683.

Blakesley, R.W., Dodgson, J.B., Mes. I. F. and Wells, R.D. (1977) J. Biol.

Chem., 252, 7300.

Boliver, R., Rodriguez, R.L., Betlach, M.C. and Boyer H.W. (1977a) Gene, 2, 75.

Boliver, R., Rodriguez, R.L., Betlach, M.C, Hevneke H.L., Boyer H.W., Closa,

L.H. and Falkow, G. (1977b) Gene, 2, 93.

Bottermann, J.H. and Zabeau, M. (1985) Gene, 37, 229.

Bowers, L.D. (1986) Anal Chem., 58,513.

Boyer, H.W. (1971) Ann. Rev. Microbiol., 25, 153.

Brandes, L., Wu, X., Bode, J., Rhee, J. I. and Schugrel, K. (1993) Biotechnol.

Bioeng., 42, 205.

Brock, T. D. (1985) Science, 230,132.

Bron, S. and Horz, W. (1980) Methods in Enzymol., 65, 112.

Brook, J.E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Iver, C.C,

Mazzola, L.M., Slatko, B.E. and Benner, J.S. (1991) Nucleic Acid Res., 19, 841.

Brooke, J.E., Berner, J.S., Heiter, D.F., Silber, K.R., Sznyter, L.A., Jaqer

Ouinton, T., Moran, L.S., Slalko, B.E., Wilson, G.G. and Nwankwo, D.O. (1989)

Nucleic Acid Res., 17, 979.

• Burnett, T.C. and Schmidt, L.L.A. (1921) J. Immunol., 6, 255.

• Burton, W.G., Robert, R.J., Myers, P.A. and Sugar, R. (1977) Proc. Natl. Acad.

Sci. USA., 74, 2687.

109

Campbell, D.H. and Fourt, I. (1939) J. Biol. Chem., 129, 385.

Carlsson, J., Mosbach, K. and BuUow, L. (1996) Biotechnol. Bioeng., 51, 221.

Carr, P.W. and Bower, L.D. (1980) In: Immobilized Enzymes in Analytical and

Clinical Chemistry. J. Wiley and Sons. New York.

Cashion, P., Javed, A., Harrison, D., Seeley, J., Zentini, V. and Sathe, G. (1982a)

Biotechnol. Bioeng., 24, 403.

Cashion, P., Lentini, V., Harrison, D. and Javed, A. (1982b) Biotechnol. Bioeng.,

24, 1221.

Catty, D. and Raykundalia, C. (1988) In: Antibodies. A Practical Approach,

(Catty, D. ed.) IRL Press, 1, 50.

Chang, TMS. (1977) In: Biomedical Applications of Immobilized Enzymes and

Protein, Plenum press. New York, 1 and 2.

Chibata, I., Tosa, T. and Sato, T. (1986) J. Molecular Catalysis, 37,1.

Cinader, B. (1967) In: Antibodies to Biologically Active Molecules (Cinader, B.

ed.) Pergamen Press, Oxford. 85.

Clark, D.S. (1994) TIBTECH, 12, 439.

Clarke, CM. and Hartley, B.S. (1979) Biochem. J., 177, 49.

Coulet, P.R., Carlsson, J. and Porath, J. (1981) Biotechnol Bioeng., 23, 663.

Davies, D.R., Sheriff, S. and Pardon, E.A (1985) J. Biol. Chem., 263, 10541.

de-Alwis, W.U. and Wilson, G.S. (1989) Talanta, 112, 249.

de-Alwis, W.U., Hill, B.S., Meiklezohn, B.I. and Wilson G.S. (1987) Anal.

Chem., 59, 2688.

Dickerson, R.E and Drew, H.R. (1981) J. Mol. Biol., 149, 761.

Dickmann, S. and McLanghlim, L.W. (1988) J. Mol. Biol., 202, 823.

Dorner, L.F. and Schildraut, I. (1994) 22, 1068.

110

• Doubradi, V., Hjdu, J., Bot, G. and Friedrich, P. (1980) Biochemistry, 19, 2295.

• Dubey, A.K., Bisaria, V.S., Mukhopadhjay, S.N. and Ghose, T.K. (1989)

Biotechnol. Bioeng., 33, 1311.

• Dubey, A.K., Mukhopadhay, S.N., Bisaria, V.S. and Ghose, T.K. (1987) Proc.

Biochem., 22, 25.

• Ehbrecht, H.J., Pingoud, A., Urbanke, C , Mass, G. and Gualerzi, G. (1985) J.

Biol. Chem., 260, 6160.

• El Rassi, Z., Truei, Y., Maa, Y.H. and Horvath, C. (1988) Anal. Biochem., 169,

1722.

• Elhe, H. and Horn, A. (1990) Bioseperations, 1, 97.

• Ernst-Cabera, K. and Wilchek, M. (1988) Trends Anal. Chem., 7, 58.

• Fahey, J.L. and Terry, E.W (1979) In: Handbook of Experimental Immunology

(Weired, D.M. ed.) Oxford Blakewell Scientific publications U.K., 1, 8.1.

• Feizi, T. and Childs, R.A. (1990) Biochem. J., 245,1.

• Fleminger, G., Hadas, F., Wolf, T. and Soloman, B. (1990) Appl. Biochem.

Biotechnol., 23, 123.

• Frederick, C.A., Grable, J., Melia, M., Samudzei, C., JenJacobson, L. Wang,

B.L., Greene, P., Boyer, H.W. and Rosenberg, J.M (1984) Nature, 309, 327.

• Fusek, M., Turkora, J., Stovickova, J. and Franek, F. (1988) Biotechnol. Lett., 10,

85.

• Gemeiner, P., Rexova-Berkova, L., Suee, F. and Narrlow, D. (1994) In:

Immobilized Biosystems. Theory and Practical Applications. (Veliky, LA. and

McLean, R.J.C. eds.) Blockie Academic and Professional. Glascow. 1.

• Gersten, D.M. and Marchalonis, J.J. (1978) J. Immunol Methods, 24, 305.

• Gestrelius, S., Mattiason, B. and Mosbach, K. (1972) Biochim Biophys. Acta.,

276, 339.

I l l

Coding, J.R. (1983) In: Monoclonal Antibodies: Principles and Practices,

Academic Press, New York, 188.

Goff, S.P. and Berg, P. (1977) In: Recombinant-Molecules, Impact on Science

and Society (Beers, R.E and Basselt, E.G. eds.) Reven, New York, 285.

Grabowski, G., Maass, G. and Alves, J. (1996) FEBS letters, 381,106.

Greene, P.J., Betlach, M.C., Goodman, H.M., and Boyer, H.W. (1974) Methods

Mol. Biol., 7, 87.

Greene, P.J., Heyneker H.L., Boliver, P., et al. (1978) Nucl. Acid Res., 5, 2273.

Greene, P.J., Ponnian, N.S., Nussbaum, A.L., Tobias, L., Gartfin, D.E., Boyer,

H.W. and Goodman, H.M. (1975) J. Mol. Biol., 99, 237.

Gronman, E.V. and Wilcheck, M. (1987) Trends Biotechnol., 5, 220.

Hale, J.E. (1995) Anal. Biochem., 231, 46.

Hale, J.E. and Beidler, D.E. (1994) Anal. Biochem., 222, 29.

Hansen, R.S. and Beavo, J.A. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 2788.

Hedgpeth, J., Goodman, H.M. and Boyer, H.W. (1972) Proc. Natl. Acad. Sci.

USA., 69, 3448.

Heitman, J. (1992) BioEssays, 14, 445.

Hemdan, E.S., Zaho, Y., Sulkowiski, E. and Porath, J. (1989) Proc. Natl. Sci.

USA., 86,1811.

Hershfield, V., Boyer, H.W., Yanofsy, C., Lovett, M.A. and Helinski, D.R.

(1974) Proc. Natl. Acad. Sci. USA., 71, 3455.

Hinch, B. and Kula, M.R. (1980) Nucleic Acid Res., 8, 623.

Hoffmann, W.L. and O'Shannessy, D.J. (1988) J. Immunol. Methods, 112, 113.

Horiuchi, K. and Zinder, N.D. (1975) Proc. Natl. Acad. Sci. USA.,72, 2555.

Horowitz, P. and Bowman, S. (1987) J. Biol. Chem. 262, 5587.

112

Hsu, M. and Berg, P. (1978) Biochemistry, 17, 131.

Ikura, K., Okumura, K., Yashikawa, M., Sasaki, K. and Chiba, H. (1984) J. Appl.

Biocliem., 6, 222.

Jack, W.E., Greenough, L., Dorner, L.F., Xu, S-Y., Strzelecka, T., Aggarwal,

A.K. and Schildkraut, I. (1991) Nucleic Acid Res., 19,1825.

Jack, W.E., Terry, B. J. and Modrich, P. (1982) Proc. Natl. Acad. Sci. USA., 79,

4010.

Jackson, D.A., Symons, R.H. and Berg, P. (1972) Proc. Natl. Acad. Sci. USA.,

69, 2904.

Jafri, F. and Saleemuddin, M. (1997) Biotechnol. Bioeng., 56, 605.

Jafri, P., Hussain, S. and Saleemuddin, M. (1995) Biotechnol. Tech., 9,117.

Jafri, P., Hussain, S. and Saleemuddin, M. (1993) Biotechnol. Appl. Biochem.,

18, 401.

Jones, S. and Thornton, J..M. (1996) Proc. Natl. Acad. Sci. USA., 193, 13.

Kerft, J. and Hughes, C. (1982) In: Gene Cloning in Organisms other thanE. coli,

(Hofschneider, P.H. and Goebels, W. eds.). Springer, Heidelberg, 1.

Kim, Y., Grable, J.C, Love, R., Greene, P.J. and Rosenberg, J.M. (1990)

Science, 1307.

Klems, J. and Citri, N. (1979) Biochim. Biophys. Acta., 567, 401.

Klibanov, A.M. (1983) Science, 219, 722.

Kuwamura, M., Sakakibara, M., Watanbe, T., Kita, K., Hiraoka, N., Obayashi,

A., Takagi, M. and Yano, K. (1986) Nucleic Acid Res., 14, 1985.

Lacks, S.A. (1980) Methods in Enzymol., 65, 138.

Laemmli, U.K. (1970) Nature, 227, 680.

Lai, C.J. and Nathans, D. (1974) J. Mol. Biol., 89, 179.

113

Lane, A.N., Jenkins, T.C., Brown, T. and Neidle, S. (1991) Biochemistry, 30,

1372.

Lao, W.D. and Chen, S.Y. (1986) Scientic Sinica., 29, 947.

Landman, A.D. and Lampert, J. (1978) Biochem. J., 169, 255.

Lee, Y.H., Blackesley, R.W., Smith, L.A. and Chirikjian, J.G. (1980) Methods in

Enzymol., 65, 173.

Lee, Y.H., Blackesley, R.W., Smith, L.A. and Chirikjian, J.G. (1978) Nucleic

Acid Res., 5, 679.

Lesser, D.R., Kurpiewski, M.R., Water, T. and Connoly, B.A. (1993) Proc. Natl.

Acad. Sci. USA., 90, 7548.

Lis, H. and Sharon, N. (1993) Eur. J. Biochem., 218, 1.

Ljungquist, C , Breitholth, A., Brink-Nilsson, H., Moks, T., Uhlen, M. and

Nilsson, B. (1989) Eur. J. Biochem., 186, 563.

Lobban, P.E. and Kaiser, A.D. (1973) J. Mol. Biol., 78, 453.

Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol.

Chem., 193, 265.

Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) In: Molecular Cloning. Cold

Spring Harbour Laboratory, Cold Spring Harbour, New York.

Martinek, K. and Mazhev, V.V. (1985) Adv. Enzymol., 57, 179.

Maschake, H.E. and Friehs, K. (1990) Production of EcoKL with different

expression plasmids in multi-plasmid-systems in E. coli. (Christiansen, C. et al.,

eds). Proc.5"' Eur. Congr. Biotechnology, Copenhagen. Munksgaard International

Publisher, Copenhagen, 163.

Maschke, H.E., Kumar, P.K.R., Geiger, R. and Schugerl, K. (1992) J.

Biotechnol., 24, 235.

114

McKenzie, G.H., Sawer, W.H. and Nichol, L.W. (1972) Biochem. Biophys Acta.,

263, 283.

McLaren, A.D. and Packer, E. (1970) Adv. Enzym. Relat. Areas. Mol. Biol., 33,

245.

Melchers, F. and Messer, W. (1970) Biochem. Biophys. Res. Commun., 40, 570.

Mertz, J.E. and Davis, R.W. (1972) Proc. Natl. Acad. Sci. USA., 69, 3370.

Micheali, J.D., Pinto, E., Benjamani, T. and de-Buren, P.P. (1969)

Immunochemistry, 6, 101.

Modrich, P. (1979) Q. Rev. Biophys., 12, 315.

Modrich, P. (1982) In CRC Crit. Rev. Biochem., 13, 287.

Modrich, P. and Roberts, R.J. (1982) In: Nucleases (Linn, S.M. and Roberts, R.J.

eds.) Cold Spring Harbour Laboratory, Coldspring Harbour, New York, 109.

Modrich, P. and Zabel, D. (1976) J. Biol. Chem., 251, 5866.

Mosbach, K. (1987) Methods inEnzymol., 135, 136.

Mosbach, K. and Mattiason, B. (1976) Methods inEnzymol., 44, 453.

Murray, N.E. and Murray, K. (1974) Nature, 251, 476.

Nardone, G. and Chirikijian, J.G. (1987) In: Gene Amplification and Analysis

(Chirikijian, J. ed.) 5, 147.

Nasri, M. and Thomas, D. (1987) Appl. Biochem. Biotechnol., 15,119.

Nerdal, W., Hare, D.R. and Reid, B.R. (1989) Biochemistry, 28,10008.

Newman, M.N., Strzelecka, T., Dorner, L.F., Schildkraut, I. and Aggarwal, A.K.

(1994) Nature, 368, 660.

Nilson, K. and Mosbach, K. (1980) Eur. J. Biochem., 112, 397.

Nilson, K. and Mosbach, K. (1981) Biochem. Biophys. Res. Commun., 102, 449.

Nilsson, B., Abrahmsen, L. and Uhlen, M. (1985) Embo. J., 4, 107.

115

• O'Shannessey, D.J. and Wilchek, M. (1990) Anal. Biochem., 191,1.

• Olszwski, J. and Wasserman, B.P. (1986) Appl. Biochem. Biotechnol., 13, 29.

• Ong, F., Gilkes, N.R., Miller, R.C., Warren, R-Jr. and Kilburn, D.C. (1991)

Enzyme Microb. Technol., 13, 59.

• Oucterlony, O. (1949) Acta. Pathol. Microbial Scand., 26, 507.

• Pepper, D.S. (1992) Methods Mol. Biol., 11, 173.

• Pingoud, A. and Jeltsch, A. (1997) Eur. J. Biochem., 240,1.

• Pitcher, W.H. (1980) In: Immobilized Enzymes for Food Processing. CRC Press

Inc. Boca Raton, Florida.

• Pleczar, M.J., Chan, E.C.S. and Krieg, N.R. (1986) In: Microbiology

International Student edition V. McGraw Hill, Singapore.

• Polisky, B., Greene, P., Garfin, D. E., McCarty, B.J., Goodman, H.M. and Boyer,

H.W, (1975) Proc. Natl. Acad. Sci .USA., 72, 3310.

• Pollock, M.R., Fleming, J. and Petrie, S. (1967) In: Antibodies to Biologically

Active Molecules (Cinader, B. ed.) 132. Pergomen Press, Oxford, 132.

• Porath, J., Axen, R. and Ernback, S. (1967) Nature, 215, 1491.

• Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. (1975) Nature, 258, 598.

• Prisyazhnoy, V.S., Fusek, M. and Alkhov, B. (1988) J. Chromatogr., 424, 243.

• Quash, G., Roch, M., Nivlean, G.J., Kevlouangknot, T. and Huppert, J. (1978) J.

Immunol. Methods., 221,165.

• Rhee, J.I., Bode, J., Diaz-Ricci, J.C, Pook, D., Wagel, B., Kretzmer, G. and

Schugerl, K. (1997) J. Biotechnol, 55, 69.

• Rhee, J. I., Diaz-Ricci, J.C., Bode, J. and Schugerl, K. (1994) Biotechnol. Lett.,

11, 225.

• Richards, F. M. and Knowles, J.R. (1968) J. Mol. Biol., 37, 231.

116

• Roberts, R.J. and Halford, S.E. (1993) In: Nuclease (Linn, S.M., Lloyd, R.S. and

Roberts, R.J. eds.) Cold Spring Harbour Laboratory Press. Cold Spring Harbour,

New York, 35.

• Roberts, R.J. and Macelis, D. (1993) Nucleic Acid Res., 24, 3125.

• Roberts, R.J., Wilson, G.A. and Young, F.E. (1977) Nature (London) 265, 82.

• Rosenberg, J.M. (1991) Curr. Opin. Struct. Biol., 1, 114.

• Roueff, P., Lillo, C. and Campbell, W.A. (1989) Biochem. Biophys Res.

Commun., 161, 496.

• Sadana, A. and Madgula, A. (1993) Biotechnol. Prog., 9, 259.

• Saha, B.C., Mathupala, S.P. and Zeikus, J. (1988) Bichem. J., 252, 343.

• Saleemuddin, M. (1999) In: Advances in Biochemical Engineering/

Biotechnology, 64, 204.

• Sambrook, J., Fritis, E.F. and Maniates, T. (1989) In: Molecular Cloning. Cold

Spring Harbour Laboratory Press, Cold Spring, USA.

• Sassenfeld, H.M. (1990) TIBTECH, 8, 88.

• Sato, P.H. and Walton, D.M. (1983) Arch. Biochem. Biophys., 221, 543.

• Shami, E.Y., Ramjeesingh, M., Rothstein, A. and Zywulko, M. (1991) Enz.

Microbiol. Technol., 13, 424.

• Shami, E.Y., Rothstein, A. and Ramjeesingh, M. (1989) Trends Biotechnol, 7,

186.

• Sheriff, S., Silverton, E.W., Padlon, E.A., Cohen, G.H., Smith, G.L., Finzel, B.C.

and Davies, D.R. (1987) Proc. Natl. Acad. Sci. USA., 84, 8075.

• Shipigel, E., Goldlust, A., Efroni, G., Avraham, A., Eshel, A., Dekel, M. and

Shoseyov, O. (1999) Biotechnol. Bioengg. 65, 17.

• Siemann, M., Syldatk, C. and Wagner, F. (1994) Biotechnol. Lett., 16, 349.

117

Silman, T.H. and Katchalski, K. (1986) Ann. Rev. Biochem., 35, 873.

Singh, P. (1984) Appl. Biochem. and Biotech., 9, 161.

Sinha, N.K. and Light, J.K. (1975) J. Biol. Chem., 250, 8624.

Smith, H.O. (1979) Science, 205, 455.

Smith, H.O. and Chirikijian, J.G. (1979) J. Biol. Chem., 254, 1003.

Smith, H.O. and Nathans, D. (1973) J. Mol. Biol., 81, 419.

Solomon, B., Hollander, Z., Koppel, R. and Katchalski-katzir, E. (1987) Methods

in Enzymol., 135,160.

Solomon, B., Koppel, R., Pines, G. and Katchalaski-Katzir, E. (1986) Biotechnol.

Bioeng., 28, 1213.

Solomon, B., Koppel, R., Schwartz, F. and Flemminger, G. (1990) J.

Chromatogr., 510, 321.

Solomon, B., Moav, N., Pines, G. and Katchalski-katzir, E. (1984) Mol.

Immunol., 21,1.

Sousa, C , CehoUa, A. and deLorenzo, V. (1996) Nature Biotechnology, 14,

1017.

Stovickova, J., Franek, F. and Turkova, J. (1991) Biocatalysis, 5, 121.

Subramanian, A. and Valander, W.H. (1992) J. Chromatogr. 591, 99.

Sulkowski, E. (1989) Bio Essays, 10,170.

Sunderberg, L. and Porath, J. (1974) J. Chromatogr., 90, 87.

Suqden, B., DeTroy, B., Roberts, R.J. and Sambrook, J. (1975) Anal. Biochem.,

68, 36.

Swatsgood, H.E., Horton, A.R. and Mosbach, K. (1976) Methods in Enzymol.,

44, 453.

118

• Taylor, J.D., Goodall, A.J., Vermote, C.L. and Halford, S.E. (1990)

Biochemistry, 29, 10727.

• Terry, B.J., Jack, W.E. and Modrich, P. (1985) J. Biol. Chem., 260, 13130.

• Thomas, CM., Stalker, D.M., Guiny, D.G.B. and Helinski, D.R. (1979) In:

Plasmids of Medical, Environmental and Commercial Importance (Timmis, K.N.

and Puhler, A. eds.) 375.

• Thomas, M. and Davis, R.W. (1975) J. Mol. Biol., 91, 315.

• Tickchonenko, T.I., Karamov, E.V., Zavizion, B.A. and Naroditsky, B.S. (1978)

Gene 4,195.

• Tietz, N.W.J. (1980) Clin. Chem. Biochem., 18, 763.

• Tischer, W. and Kasche, V. (1999) TIBTECH, 17, 326.

• Turkova, J. (1993) In: J. Chromatogr. Lib., Elseivier, 55, 664.

• Turkova, J. (1999) J. Chromatogr. B. Biomed. Sci. App., 722, 11.

• Ulbrich-Hoffmann, R., Golbik, R. and Damerau, W. (1993) In: Stability and

Stabilization of enzymes, (Vander Tweel, W.J.J., Harder, A. and Buitelor, R.M.

eds.), Elsevier, 497.

• Watabe, H., Shibata, T. and Ando, T. (1981) J. Biochem., 90, 1623.

• Watabe, H., Shibata, T., Ino, T. and Ando, T. (1984) J. Biochem., 95, 1677.

• Weber, K. and Osborn, M. (1969) J. Biol. Chem., 244, 4406.

• Wells, R.D., Klein, R.D. and Singleton, C.K. (1981) In: The Enzymes (P.D.

Boyer ed.), 3rd ed., Academic Press, New York, 14, 157.

• Wieseman, A. (1969) Process Biochem., 63.

• Wieseman, A. (1980) Chem. Technol. Biotechnol., 30, 521.

• Wingard, H.D.B. and Egler, K.A. (1984) Appl. Biochem. Biotechnol., 9, 131.

• Woodhead, L. and Malcolm, A.D. (1981) Eur. J. Biochem., 120, 125.

119

mierobial technology

ELSEVIER Enzyme and Microbial Technology 25 (1999) 172-176

Research papers

Immobilization of the restriction endonuclease EcoRl Usefulness of a polyclonal antibody support

Hemlata Varshney^ Jawaid IqbaP, Mohammed Saleemuddin''''''*

'Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, U.P., India ^Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, U.P.. India

Received 8 June 1998; accepted 10 January 1999

Abstract

The restriction enzyme EcoRl has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the y-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B. was highly effective in binding EcoRl from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRl immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear A-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of £«;RI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65°C. It was also possible to immobilize EcoRl directly from the cell lysates of Escherichia call pMB4, an £coRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRl to the antibody support. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Restriction enzymes; Enzyme stabilization; Immunoaffinity immobilization

1. Introduction

Nucleases in general and restriction endonucleases (REs) in particular are widely used in several areas of nucleic acid research including recombinant DNA technology. Type II REs are routinely used in the physical mapping of DNA, plasmid construction, gene cloning, cDNA construction, etc. [1] The high cost of REs and their lability, however, add remarkably to the cost of such investigations. Strategies that stabilize REs and facilitate their recovery and reuse have, therefore, received significant attention.

EcoRl and BamWl were immobilized on CNBr-activated Sepharose [2], and along with Hindlll on trityl agarose [3]. Limitations of the procedures prompted Wingard and Egler [4] to couple lysine to the carboxyl groups of EcoRl, BamHl, and Bgll as a handle for immobilization on supports reactive toward amino groups. Most of the REs investigated, however, exhibited sensitivity to modification of both

* Corresponding author. Fax: +91-571 -401 -081. E-mail address: [email protected] (M. Saleemuddin)

carboxyl and amino groups that are usually used in the coupling of enzymes to solid supports [5,6].

Entrapment procedures that do not result in chemical modification of enzyme have also been investigated for the immobilization of EcoRl and Pstl [5]. Taking the large dimensions of DNA substrates into consideration, highly porous polymeric matrices are required for effective en-zyme-DNA contact, but such matrices also have the inherent risk of enzyme leaching. Crosslinking of the entrapped enzymes with appropriate bifunctional reagents may restrict the leaching of the entrapped enzymes but not without causing chemical modification of their amino acid side-chain groups [5].

One immobilization strategy that does not result in the chemical modification of the enzymes but may yield highly active and stable preparations involves the use of immunoaffinity supports. Both monoclonal and polyclonal antibody supports have been shown to yield highly active preparations because of the antibodies acting as large spacer between the enzyme and support [7,8]. We have investigated the utility of Sepharose-bound polyclonal anti-fcoRI antibody supports in the immobilization and stabilization of the enzyme and its direct immobilization from the lysate of an

0141-0229/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PlI: 80141-0229(99)00030-7

mailto:[email protected]

H. Varshney et al. /Enzyme and Microbial Technology 25 (1999) 172-176 173

EcoRl overproducing Escherichia coli strain. The results of the study are described in this manuscript.

2. Materials and methods

EcoRI (EC 3.1.23.13) from E. coli RY13, the plasmid pBR322, and A-DNA were obtained from Genei Laboratories, Bangalore, India. The plasmid pBHLUC P, a 7.032-kb, 5' deletion plasmid of pBHLUC 1.3 containing a single EcoRl site, was kindly supplied by Dr. A. Rahman of the University of Chicago Department of Pharmacology. Agarose type III used for electrophoresis was the product of Sigma Chemical Co., E. coli pMB4, an EcoRl overproducing strain was kindly provided by Prof. H.K. Das of Jawa-harlal Nehru University, New Delhi, India.

The procedure described by Lee et al. [2] was followed for the determination of £coRI activity. The reaction mixture contained in a total volume of 50 JLII, 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgClj, 10 mM 2-mercap-toethanol, 1 jxg of DNA, and appropriate quantity of soluble or immobilized enzyme. The mixture was incubated, unless mentioned otherwise, for 1 h with slow but continuous agitation and reaction terminated by the addition of the dye reagent (0.2% SDS, 70% sucrose, 0.1% bromophenol blue).

Agarose gel electrophoresis of native and EcoRI treated DNA or the plasmids pBR322 and pBHLUC P was performed in a horizontal slab gel apparatus. The gels were cast with 1% agarose and subjected to electrophoresis in TAE buffer (Tris 40 mM, 24 mM acetic acid, 0.9 mM EDTA, pH 8.5) at constant current of 50 V. The gels were stained with ethidium bromide in distilled HjO overnight and photographed by illumination under UV light.

Rabbits weighing 1.5-2.0 kg were subcutaneously injected with emulsion prepared by mixing 95 ju,l of Freund's complete adjuvant, 400 /ng EcoRI, and 300 ju-l distilled water. Pre-immunized sera were drawn from the rabbits and used as control. Animals received 400-/xg doses of EcoRl along with the adjuvant weekly over a period of 4 weeks. The animals were rested for 2 weeks and administered booster dose of 400 /ng of emulsified enzyme. The rabbits were bled by cardiac puncture, clot formation allowed for 3-4 h at room temperature, and the samples centrifuged at low speed to separate the serum from the clot. The sera were decomplemented at 56°C for 30 min, mixed with sodium azide, quickly frozen, and stored at -20°C until further use.

The antigen-antibody reaction was carried out by the Ouchterlony double diffusion method [9] in agarose slabs. Ammonium sulfate fractionation of the immunized sera for partial purification of the y-globulin fraction was carried out, and fractions of 20-40% saturation were collected [10]. The fraction was dialyzed and coupled to Sepharose 4B [11]. The preparation used as immunoaffinity support possessed 20.7 mg of protein per g of Sepharose. Amounts of protein coupled were determined by subtracting from the

Fig. 1. immunodiffusion of anti-EcoRI antiserum against EcoRl. Immunodiffusion was carried out in 1% agarose slabs as described in the text. The central well contained 280 U of EcoRl while 28 /nl of the antiserum was placed in the peripheral wells.

amount of that added those of the supematent and washings [12].

Immobilization of EcoRI was performed by incubating 250 il of the immunoaffinity support with 200 U of EcoRl in a total volume of 500 /il containing 0.5 mM EDTA, 50% glycerol, 10 mM phosphate buffer, pH 7.6, at 4°C for 5 h. The matrix was separated from the unbound enzyme by centrifugation and washed with a solution of 0.5 mM NaCl, 25 mM Tris-HCl, pH 7.5, 10% glycerol and 1 mM DTT. One unit of enzyme is the amount needed to digest 1 /j,g of A-DNA in 1 h at 37°C under the conditions.

The E. coli pMB4 was grown overnight in nutrient broth containing 10 jLtg/ml ampicillin [13,14]. The cells were collected by centrifugation and the pellet suspended in a minimum volume of 1 ml of TA buffer (50 mM Tris-HCl. pH 7.6; 30 mM NaCl). The suspension was mixed with 5 ml of lysis buffer (50 mM Tris-HCl pH 7.6, 30 mM NaCl, 11.2 mM EDTA, 40 /xg/ml lysozyme and 40% w/v sucrose) and the sample incubated at 0°C overnight. The debris and unlysed cells were centrifuged at 12 000 X g and the clear supernatant used as crude homogenate. For the immobilization of EcoRI from the lysate, 250 ^1 of the immunoaffinity support was mixed with 250 /oil of E. coli pMB4 lysate and incubated for 5 h at 4°C. The support matrix was isolated by centrifugation and washed with the buffer several fimes.

3. Results and discussion

Rabbits injected with EcoRI along with the adjuvant readily responded by producing antibodies, and the antisera gave a single sharp precipifin line on immunodiffusion against EcoRI (Fig. 1). The antibodies were not inhibitory toward EcoRI even when added in excess, and effective immunoadsorbant could be prepared by coupling the 7-globulin fraction isolated from the serum of immunized

174 H. Varslmey el al. /Enzyme and Microbial Technolofiy 25 (1999) 172-176

Lane 1 2 3 A 5 6 7

Fig. 2. fmmobifization of EcoRl on the immunoaffinity support. Digestion of A-DNA was carried out using soluble or immunoaffinity immobilized EcoR] as described in the text. One |ig of DNA was incubated with either 4 U of soluble EcoRl (Lane 2). . 0 p.1 of immobilized EcoRl (Lanes 3, 4, and 5). 30 /J.1 supernatant obtained after incubation of froRl with the immunoaffinity support (Lane 6) or 30 ij.\ of the first wash of the enzyme after the immobilization process (Lane 7). Lane 1 contained A-DNA not exposed to either soluble or immobilized £(Y)RL

Lane 1 5 6

Fig. 3. Digestion of the plasmid pBR322 and pBHLUC P by soluble and antibody support-immobilized EcoRl. 0.5 /j.g of pBR322 or 0.5 /xg. Soluble or immobilized preparations of fcoRI (4 U) were incubated with 0.5 /Lig of the plasmids under .standard conditions and the digests subjected to electrophoresis. Lane 1. pBR322 alone. Lane 2, pBR322+ soluble EcoRl. Lane 3, pBR322+ immobilized EcoRl. Lane 4. pBHLUC P alone. Lane 5, pBHLUC P soluble EcoRl. Lane 6, pBHLUC P+ immobilized EcoRl.

rabbit to CNBr-activated Sepharose-4B. Some plausible explanations offered for the absence of inhibitory antibodies in the antisera include the active site acting as blind spot for the immune system and steric hindrance caused by the binding of high affinity antibodies recognizing adjacent locations of the active sites [15].

Each 100 /j,l of matrix bearing 20.7 mg/ml y-globulin fraction of anti-EcoRI antiserum bound approximately 225 U of EcoRl under the conditions used in the study as detailed in the text. Only about 50% of the bound activity of the EcoRl could be observed on the matrix by using A-DNA as the substrate. Enzymes bound to antibody supports have been generally shown to exhibit high activity, presumably due to the antibody molecules acting as large spacer [7,15, 16]. The observed lower activity of the bound EcoRl, despite the noninhibitory nature of the antibodies, may be related to steric hindrance due to the very large molecular weight of the DNA used as substrate. It is also likely that as a result of the random coupling of the antibodies via their amino groups to the CNBr-activated supports, some of the immobilized antibody molecules may bind EcoRl. molecules in orientation that restricts their access to the large molecular weight DNA. Other molecules of the enzyme may be favourably oriented and fully accessible to the DNA substrate, as evident from the fragmentation pattern of the DNA which was identical to that obtained with the soluble EcoRl (Fig. 2). Lack of alteration of cleavage specificity of immunoaffinity-bound EcoRl was not restricted to A~DNA alone. Fig. 3 shows that the action of both the soluble and the immobilized EcoRl was identical on the coiled/super-coiled plasmids pBR322 and pBHLUC P. In either case, both the coiled and supercoiled forms were transformed to the linear forms because of the restriction activity.

The EcoRl bound to the antibody support also exhibited

a clear improvement in stability against heat-induced inac-tivation (Fig. 4). While the native enzyme lost nearly all restriction activity on incubation at 45°C, that immobilized on the antibody support retained nearly unaltered activity even after incubation at 55°C. Preparations incubated even at 60°C or 65 °C also showed low but significant restriction activity. Because the EcoRl preparations were exposed to various temperatures and cooled before assay under standard conditions, the observed stability may also be related, at least in part, to the ability of the immobilized enzyme to regain activity after thermal denaturation. Immobilization has been shown to facilitate protein refolding after denaturation by restricting protein-protein aggregation [17,18].

Both polyclonal [8,19-21] and monoclonal [22-24] antibody supports have been shown to be effective in enhancing the resistance to denaturation of a number of enzymes. Antibody binding appears to involve reasonably large areas of protein antigen which may comprise of more than one polypeptide chain, and the stability enhancement may consequently arise out of a crosslinking-like effect [25,26]. Some antibodies may confer remarkable stability if they are directed toward and bind to regions of enzyme that may be crucial in the initiation of the unfolding process [27,28]. The antibody support-associated EcoRl was also stable to storage at 4°C for several weeks and could be reused several times (data not included).

In view of the high degree of specificity of interaction between antibody and antigen, the feasibility of using the immunoaffinity adsorbent for directly immobilizing EcoRl from a crude preparation was investigated. For this purpose, the antibody-support was incubated with lysate of E. coli pMB4, a strain that overproduces EcoRl. The matrix was washed and incubated with DNA, and as shown in Fig. 5, fragmentation pattern of DNA by the immobilized preparation was identical with that produced by soluble EcoRl.

H. Varshney et al. I Enzyme and Microbial Technology 25 il999) 172-176 175

Lane 1 8

Fig. 4, Effect of pretreatment at various tempieratures on soluble (A) and antibody support-immobilized (B) preparations of iEcoRl. In a total volume of 50 /xl, 4 U of soluble or immobilized £coRI were exposed to various temperatures for 15 min, cooled for 30 min at 0°C and restriction activity determined on A-DNA under standard conditions. Lane 2 contained EcoRl sample not subjected to the heat treatment, while those in Lanes 3, 4, 5, 6, 7, and 8 contained samples exposed to 37°C, 45°C, 50°C, 55°C, 60°C, or 65°C, respectively. Lane 1 contained A-DNA.

More interestingly, the nonspecific digestion of the DNA that is strikingly evident after incubation of lysate with the DNA was clearly absent in the samples incubated with the immobilized enzyme. This indicates specificity in the binding of EcoRl onto the antibody support. The possibility of nonspecific association of some lysate proteins not related to DNA cleavage with the support cannot be ruled out, but it should be possible to minimize such association using highly purified/monoclonal antibodies. Other instances of the use of polyclonal/monoclonal antibody support in the direct immobilization enzymes from cell lysates are also available [29,30].

This study shows the remarkable potential of using the anti-£coRI antibody support in the stable immobilization of the restriction enzyme EcoRl also from the crude lysates of an £coRI-overproducing strain of E. coli. While it may be possible to improve the performance of the immobilized enzyme by replacing the polyclonal antibodies with appropriate monoclonal antibody, the former, if noninhibitory as in the present study, may offer the advantage of lower cost and higher stability [31]. Interesting strategies for raising

Lane 1 2 3 A 5 6 7

Fig. 5. Time-dependent cleavage of DNA by E. coli pMB4 lysates and the immobilized preparation obtained by incubating the anti-EcoRI antibody Sepharose with the lysates. Details of preparation of lysates and incubation with the support are given in the text. Aliquots lysates of E. coli pMB4 or those containing the immobilized preparation were incubated with A-DNA for various indicated intervals under standard conditions. Lane 1 contained A-DNA alone while DNA incubated with 4 U of EcoRI was loaded in Lane 2. Lanes 3, 4, 5, 6, and 7 contained the A-DNA incubated for 10, 15. 30, 45 or 60 min, respectively, with the lysate (A) or the immobilized preparation (4 U) (B).

noninhibitory polyclonal antienzyme antibodies in animals are also available [32-34].

Acknowledgment

Council of Scientific and Industrial Research (India) provided financial assistance in the form of a research grant and Junior Research Fellowship to H.V. Suggestions and help from Ms. Nilofer S. Khan are acknowledged.

References

[1) Wells RD, Kelein RD, Singleton CK. Type II restriction enzymes. Enzymes 1981;15:157-91.

[2] Lee YH, Blackesley RW, Smith LA, Chirikjian JG. Preparation and properties of insolubilized restriction endonucleases. NucI Acid Res 1978;5:679-89.

[3] Ca.shion P, Javed A, Harrison D, Seeley J. Lentini V, Sathe G. Enzyme immobilization on tritylagarose. Biotechnol Bioeng I982;24: 403-23.

176 H. Varshney et al, /Enzyme and Microbial Technology 25 (1999) 172-176

[4] Wingard LB Jr, Egler K. Covalent attachment of lysines to commercial restiiction endonucleases. AppI Biochem Biotechnol 1984;9: 131-42.

[51 Singh P. Immobilization of the restriction endonuclease Pst\. Appl Biochem Biotechnol 1984;9; 161-72.

[6| Nasri M, Thomas D. Immobilization of the re.striction endonucleases PuvII and Hindlll. Appl Biochem Biotechnol 1987;15:119-29.

[7] Solomon B. Koppel R, Pines G, Katchalski-Katzir E. Enzyme immobilization via monoclonal antibodies. Preparation of a highly active carboxy peptidase A. Biotechnol Bioeng 1986;28:1213-21.

(81 Jafri F, Husain S, Saleemuddin M. Immobilization and stabilization of invertase using specific polyclonal antibodies. 1993;18:401-8.

[9] Ouchterlony O. Antigen-antibody reactions in gels. Acta Pathol Microbiol Scand 1949;26:507-10.

1101 Fahey JL, Terry EW. Ion exchange chromatography and gel filtration. In: Weired DM, editor. Handbook of experimental immunology. Oxford, UK: Blackwell Scientific Publications, 1979. p. 8.1-8.6.

I l l ] Porath J, Axen R, Emback S. Chemical coupling of proteins to agarose. Nature (Lond) 1967;215:1419-92.

[12] Lowry OH, Rosenbrough NJ, Farr AL, Randall. Protein measurements with Folin-phenol reagent. J Biol. Chem 1951;193:265-75.

[13] Miahara M, Mise K. Rapid method for detection of restriction endo-nuclease-producing strain in endopathogenic bacteria. Anal Chim Acta 1988;213:273-7.

[14] Green PJ, Heyneker HL. Bolivar F, et al. A general method for the purification of restriction enzymes. Nucl Acid Res 1978;5:2273-82.

[15] Shami EY, Rothstein A, Ramjeesingh M. Stabilization of biologically active proteins. Trends Biotechnol 1989:7:186-90.

[16] Shami EY, Ramjeesingh M, Rothstein A, Zyulko M. Stabilization of enzymes by their antibodies. Enzyme Microb Technol 1991;13:424-9.

[17] Sinha NK, Light JA. Refolding of reduced denaturated trypsinogen and trypsin immobilized on agarose beads. J Biol Chem 1975:250: 8624-89.

[18] Horowitz P, Bowman S. Reversible thermal denaturation of immobilized rhodonase ] Biol Chem 1987;262:5587-91.

[19] Jafri F, Husain S, Saleemuddin M. Stabilization of yeast invertase by insoluble immune complex formation using unfractionated, glycosyl-specific and peptide-specific polyclonal antibodies. Biotechnol Tech 1995;9:117-22.

[20] Sato PH, Walton DM. Glutaraldehyde-reacted immunoprecipitates of L-gulonolactone oxidase are suitable for administration to guinea pigs. Arch Biochem Biophys 1983:221:543-57.

[21 ] Siemann M, Syldatk C, Wagner F. Enhanced stability of an L-hydantoinase by its corresponding polyclonal antibody. Biotechnol Lett 1994;16:349-54.

[22] Melchers F, Messer W. Enhanced stability against heat denaturation off. coli wild type and mutant B-galactosidase in presence of specific antibodies. Biochem Biophys Res Commun 1970:40:570-5.

123] Rouff P, Lillo C, Campbell WH. NADH .substrate inhibition and enhanced thermal stability of nitrate reductase immobilized via monoclonal antibody. Biochem Biophys Res Commun 1989:161:496.

[24] Solomon B, Hollander Z, Koppel R, Katchalski-Katzir E. Use of monoclonal antibodies for the preparation of highly active immobilized enzymes. Methods Enzymol 1987;135:160-70.

[251 Sheriff S, Silverton EW, Padlon EA, et al. Three-dimensional .structure of an antibody-antigen complex. Proc Natl Acad Sci USA 1987; 84:8075-9.

[26] Jones S, Thornton JM. Principles of protein-protein interaction. Proc Natl Acad Sci USA 1996;193:13-20.

[27| Arnold U, Rucknagel KP, Schierhorn A, Ulbrich-Hofmann R. Thermal unfolding and proteolytic susceptibility of ribonuclease A. Eur J Biochem 1996;237:862-9.

(28[ Ulbrich-Hoffmann R, Golbik R. Damerau W. In: Van der Tweel WJJ, Harder A, Buitelor RM, editors. Stability and stabilization of enzymes. Amsterdam: Elsevier, 1993:497-504.

[29] Agnellini D, Pace M, Cenquanta S, Gardana C, Pietta PG, Mauri PL. Characteristics of bioreactors made with urease and NAD glycohy-drolase reversibly bound to immobilized antibodies. Biocatalysis 1992;6:251-6.

[30[ Ikura K, Okumura K, Yoshikawa, M. Sasaki R, Chiba H. Specific immobilization of an enzyme by monoclonal antibody: immobilization of guinea pig liver transglutaminase, J Appl Biochem 1984;6: 222-31.

[31] deAlwis U, Wilson GS. Strategies for the reversible immobilization of enzymes by the use of biotin-bound anti-enzyme antibodies. Ta-lanta 1989:112:249.

[32] Fusek M, Turkova J, Stovickova J, Franek F. Polyclonal antichymo-trypsin antibodies suitable for oriented immobilization of chymotryp-sin. Biotechnol Lett 1988;10:85-98.

[33[ Jafri F, Saleemuddin M. Immobilization of invertase on sepharose-linked enzyme glycosyl recognizing polyclonal antibodies. Biotechnol Bioeng 1997;56:605-9.

[34] Stovickova J. Franek F, Turkova J. Polyclonal antibodies for an immobilized trypsin. Biocatalysis 1991:5:121-30.

Woodword, J. and Wiseman, A. (1978) Biochim. Biophys. Acta., 527, 8.

Xia, Y., Burbank, D.E. and Van Etten, J.L. (1986,a) Nucleic Acid Res., 14, 6017.

Xia, Y., Burbank, D.E., Uher, L., Rubussay, D. and Van Etten, L. (1986,b)

Mol. Cell Biol., 6,1430.

Xu, S.Y. and Schildkraut, I. (1991) J. Biol. Chem., 266, 4425.

Yang, S., Veide, A. and Enfors, S.O. (1995) Biotechnol. Appl. Biochem., 2, 145.

Yoshimori, R.N., RouUand, D.D. and Boyer, H.W. (1972) J. Bacteriol., 112,

1275.

Yuan, R. (1981) Annu. Rev. Biochem., 50, 285.

Zopf, D.A., Tasi, CM. and Ginsburg, V. (1978) Arch. Biochem. Biophys., 1851,

61.

Zusaman, R. and Zusaman, I. (1995) Biotechnol. Appl. Biochem., 21, 161.

120

List of Presentation/Publications

Varshney, H. and Saleemuddin, M. (1997) Immobilization and stabilization

of EcoRl on polyclonal antibody support. Presented in the 66' annual

meeting of SBC held from 22-24 December in Vishakapatnam.

Varshney, H., Iqbal, J. and Saleemuddin, M. (1999) Immobilization of the

restriction endonuclease EcoRl Usefullness of a polyclonal antibody

support, Enzym. Microb, TechnoL, 25,172-176.

Varshney, H. and Saleemuddin, M. (2000) Immobilization of BamHl on

Sepharose supports bearing randomly coupled or favorably oriented

polyclonal antibodies J. Mol. Recog. (Communicated).

BIOCHEMISTRY - CORE

Documents

Transcript of BIOCHEMISTRY - CORE