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PURIFICATION OF THREE MUTANT

CYTOCHROME C7-TYPE PROTEINS FROM

GEOBACTER SULFURREDUCENS.

ABSTRACT: Three mutant forms of periplasmic cytochrome A (PpcA) from Ge-

obacter sulfurreducens were successfully isolated and purified using cation ex-

change chromatography. Spectrophotometer analysis of the samples show evi-

dence that the mature triheme protein was present in the first peak fractions. Mu-

tant versions contain cysteine substitutions at different points of the amino acid se-

quence of PpcA. It is believed that the sulfhydryl group on the cysteine side-chain

could possibly accept a synthetic molecule that would be able to utilize the elec-

tron transfer pathway in the PpcA macromolecule. The mutants that were suc-

cessfully supplied to the collaborators at the Chemistry Division during the course

of the summer 2012 SULI project are named E39C, K29C and K70C.

I. Introduction

PpcA is a triheme protein that is found in the periplasm of Geobacter sulfurreducens. It

consists of 71 amino acids and has three heme prosthetic groups which are covalently bound to

the polypeptide chain via two cysteine thioether bonds. [1]. Mutational studies require the gene

that codes for PpcA to be heterologously overexpressed in Escherichia coli which is the most

common expression host [2]. Although E. coli does code for its own c-type cytochromes, it is

not as efficient at post-translational heme attachment as G. sulfurreducens, therefore total yields

of PpcA that are overexpressed in E. coli are relatively low [2].

II. Materials and Methods.

A. Culture growth/expression. A 50 mL flask containing 2xYT growth medium, am-

picillin (100 μg/mL) and chloramphenicol (34 μg/mL) was inoculated with a single colony of E.

coli that had already transformed with the gene that codes for PpcA. The 50 mL flask was incu-

bated overnight in a shaker at 30⁰C (250 rpm). Four 1L flasks containing 2xYT growth medium

were inoculated with 10 mL of the culture that had been grown overnight, and then incubated at

30⁰C at 250 rpm for 8-10 hours. IPTG was added to each flask to a final concentration of 100

μM and shaking was reduced to 200 rpm and culture was allowed to grow overnight.

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B. Isolation of periplasmic fraction. Cells were harvested by centrifugation with SLC-

4000 rotor for 15 min at 4000 rpm, 4⁰C. Supernatant was removed and pellet was gently re-

suspended with a rubber policeman in 40 mL TES buffer (100 mM Tris-HCl, pH 8.0, 0.5 mM

EDTA, 20% sucrose). Protease inhibitor tablet was added to the suspension. Lysozyme (0.5

mg/mL) was then added to the suspension and incubated at 4⁰C for about one hour. An equal

amount of 4⁰C milli-Q water was added and suspension was left to incubate with gentle shaking

for 15 additional minutes. Suspension was then centrifuged with SS-34 rotor at 20K rpm for 15

min, 4⁰C. The supernatant which is the periplasmic fraction and containing the protein was col-

lected and pellet was discarded.

C. Ion exchange. 1Standard protocol. Periplasmic fraction was concentrated by loading

onto a cation exchange column containing Ionosphere resin and equilibrated with 10mM Tris-

HCl pH 7.5. and then re-eluted with 10mM Tris-HCl pH 7.5 + 1 M NaCl. We then dialyzed the

prep against 100 fold excess of 10 mM Tris-HCl, pH 7.5. Sample was then centrifuged (30 min,

20K rpm, 4⁰C) and any precipitate was removed. Suspension was reloaded onto the same cation

exchange column and equilibrated with 10 mM Tris-HCl, pH 7.5 then eluted with a 150 mL gra-

dient of 0-300 mM NaCl in 10mM Tris-HCl, pH 7.5 and collected in 3 mL fractions using

FRAC-100 collector. All fractions that had an OD reading ratio A408/A210 closest to 0.8 were

considered electrophoretically pure.

1. K70C was first mixed with a 1:2 ratio with 10mM Tris-HCl, pH 7.0 buffer and centri-

fuged at 20K rpm for 15 min, 4⁰C. Periplasm was loaded onto cation exchange column packed

with Ionosphere resin at 1.2 mL/min. Protein did not bind to column and flowthrough (named

K70C.2) retained a pink color and was saved for further purification steps. Column was washed

with 10mM Tris-HCl, pH 7.0. Flowthrough was diluted further to 3:1 with 10mM Tris-HCl, pH

7.0 buffer (200 mL K70C periplasm + 400 mL Tris-HCl) and loaded onto an exchange column

containing Sp sepharose matrix. Periplasmic fraction was re-eluted using 10mM Tris-HCl, pH

7.5 + 1M NaCl. Flowthrough was then dialyzed in ~100 fold excess 10mM Tris-HCl pH 7.5

overnight. Prep was collected the following morning and centrifuged at 20K rpm for 30 min,

4⁰C. Supernatant was placed into a graduated cylinder and loaded onto a cation exchange col-

umn after diluting 2x with 10mM Tris-HCl, pH 7.5. Both K70C and K70C.2 were ultimately

loaded onto the same cation exchange column containing ~31.4 cm3 ionosphere matrix and

equilibrated with 20mM Tris-HCl, pH 8.0 + 1 mM DTT. Column was eluted with a gradient of

0-100% of buffer B using 20mM Tris-HCl, pH 8.0 + 1mM DTT for buffer A, and 20mM Tris-

HCl, pH 8.0 + 0.5M NaCl for buffer B. Fractions were collected using Frac-100 in 3 mL incre-

ments. Absorbance of the first peak was measured using Shimadzu UV160U UV-visible record-

ing spectrophotometer (Fig. 1-2).

2. E39C periplasm was first dialyzed overnight in excess 10mM Tris-HCl, pH 7.5. Cati-

on exchange column containing Ionosphere was equilibrated with 10mM Tris-HCl, pH 7.5 at 1

mL/min. Periplasm was loaded onto column at 2.0 mL/min. Column was then eluted using

1 Deviations from the Standard protocol developed by Y. Londer are explained in detail under the subheadings K70C,

E39C and K29C respectively

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10mM Tris-HCl, pH 7.5. + 0.5M NaCl at 1 mL/min. Flowthrough (~405 mL) was re-loaded on-

to exchange column and a concentration gradient using a Pharmacia P-500 pump (Run Method

#6) using 10mM Tris-HCl, pH 7.5 for buffer A and 0.5M NaCl for buffer B. Buffer B was

switched to 10mM Tris-HCl, pH 7.5. + 1M NaCl after four hours (Pharmacia pump readings:

Time/Vol = 222.8; Flow Rate= 1.0; %B= 37.0) to speed up elution process. Fractions were col-

lected in 3mL increments using Frac-100 fraction collector when Pharmacia P-500 had the fol-

lowing estimated readings: Time/Vol ~250.0mL; Flow Rate= 1.0; %B = 37<%B<45.5. Absorb-

ance of the first peak was measured using Shimadzu UV160U UV-visible recording spectropho-

tometer (Fig. 3-4).

3. K29C periplasm (~590 mL) was first dialyzed in ~100 fold excess 10mM Tris-HCl,

pH 7.5 overnight. Periplasm was collected from dialysis tubes and centrifuged for 30 min at 20K

rpm, 4⁰C. Supernatant (~900 mL) was collected and pellet discarded. Periplasm was then load-

ed onto cation exchange column containing Ionosphere resin at ~2-3 mL/min. After protein was

bound, column was washed with 1200 mL of 10mM Tris-HCl, pH 7.5. Column was then eluted

with 10mM Tris-HCl, pH 7.5 + 0.5M NaCl at 2.0 mL/min. Flowthrough (~140 mL) was dia-

lyzed overnight in ~100 fold excess 10mM Tris-HCl, pH 7.5. Suspension (~220 mL) was col-

lected from dialysis tubes and centrifuged for 30 min at 20K rpm, 4⁰C. Supernatant was loaded

onto a cation exchange column and washed with 10mM Tris-HCl, pH 7.5. An elution gradient

was done using Pharmacia P-500 pump (run method #6) using 10mM Tris-HCl, pH 7.5 for buff-

er A and 10mM Tris-HCl, pH 7.5. + 0.5M NaCl for buffer B. Protein was moving slowly

through column, so buffer B was switched to 10mM Tris-HCl, pH 7.5 + 1M NaCl. Fractions

were collected in 3 mL increments using Frac-100 fraction collector. Absorbance of the first

peak was measured using Shimadzu UV160U UV-visible recording spectrophotometer (Fig. 5-

6).

III. Results and discussion

K70C. The major peak fractions 55-59 (Fig. 1) were collected in 3 mL increments and

the A408/A210 ratio was analyzed using a Shimadzu® UV160U UV-visible recording

spectrophotometer.

Figure 1. K70C absorbtion at 408/210nm. Shows fraction #57 to have the highest heme/peptide

ratio closest to 1.0.

λ 408 210

No. A1 A2 A1/A2

55 0.375 0.795 0.4717

56 0.593 0.926 0.6405

57 0.578 0.903 0.6403

58 0.362 0.709 0.5107

59 0.188 0.56 0.3363

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Figure 2. K70C analysis of fraction #57 in the oxidized and reduced states. The α peak at 552.0

nm (A= 0.1486) and the red shifted γ peak at 418nm (A= 0.7515) are signatures that show our

triheme protein is present when the sample is reduced with sodium dithionite.

Figure 3. . E39C absorbtion at 408/210nm. Shows fraction #24 to have the highest

heme/peptide ratio closest to 1.0.

λ 408 210

No. A1 A2 A1/A2

21 0.763 1.504 0.5075

22 1.135 1.857 0.6112

23 1.625 2.154 0.7543

24 1.918 2.266 0.8466

25 1.947 2.308 0.8439

26 1.715 2.24 0.7654

27 1.436 2.169 0.6619

28 1.315 2.167 0.607

29 1.328 2.223 0.5971

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

250 400 550

Ab

so

rban

ce

λ nm

K70C Fr#57

K70C-Ox

K70C-Red

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Figure 4. E39C analysis of fraction #24 in the oxidized and reduced states. The α peak at 552.0

nm (A= 0.1750) and the red shifted γ peak at 419.5nm (A= 0.9982) are signatures that show our

triheme protein is present when the sample is reduced with sodium dithionite.

Figure 5. K29C absorbtion at 408/210nm. Shows fraction #13 to have the highest heme/peptide

ratio closest to 1.0.

λ 408 210

No. A1 A2 A1/A2

9 0.144 0.404 0.356

10 0.189 0.452 0.4176

11 0.225 0.474 0.4748

12 0.26 0.522 0.4979

13 0.254 0.509 0.4994

14 0.218 0.484 0.4501

15 0.175 0.448 0.3917

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Figure 6. K29C analysis of fraction #13 in the oxidized and reduced states. The α peak at 552.0

nm (A= 0.1882) and the red shifted γ peak at 418.0nm (A= 1.1471) are signatures that show our

triheme protein is present when the sample is reduced with sodium dithionite.

Calculations:

1.

= mg/mL

2.

An estimated amount of 1-2 mg of each mutant (K70C, E39C and K29C) was supplied to

the collaborators in the Chemistry Division after purification. Although a solution to low effi-

ciency of post-translational modifications (the gene cluster ccmABCDEFG in E. coli that is re-

sponsible for heme attachment of its own c-type cytochromes) has been discovered, this yield is

still lower than the wild type PpcA grown under the same general protocol [2]. This could have

to do with the amount of positive charge lost when lysine is substituted with cysteine. This slight

charge difference may make it more difficult for the mutants K70C and K29C to bind to the ion-

osphere matrix in the cation exchange column. As a result, some of the mature proteins contain-

ing all three prosthetic heme units may have been lost during the binding process and may also

have affected the amount of mature protein that was eluted during the concentration gradient.

Although it is not yet clear, this observation could be supported by the results since the mutant

E39C had a negatively charged glutamic acid residue replaced with a neutral cysteine and had

more affinity to the binding matrix and supplied the highest yield of protein with a peptide/heme

ratio closest to 1.0. Future purification of a new strain of K70C at a higher pH level may in-

crease its binding affinity to the column. Also by dialyzing the periplasm before trying to bind it

to the cation exchange column might get rid of some of the impurities in the periplasmic fraction

and aid in better binding of the protein.

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References

[1] P. Raj Pokkuluri, Yuri Y. Londer, Norma E.C. Duke, W. Chris Long, Marianne Schiffer,

Family of Cytochrome c7-Type Proteins from Geobacter sulfurreducens: Structure of One Cyto-

chrome c7 a1 1.45 Ḁ Resolution. Biochemistry (2004) 1-2.

[2] Yuri Y. Londer, P. Raj Pokkuluri, David M. Tiede, Marianne Schiffer, Production and pre-

liminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurre-

ducens in Escherichia coli. BBA (2002) 1-2.

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Acknowledgments:

I would like to thank the Department of Energy, all of the members of the SULI program,

Argonne National Laboratories, and especially Dr. P. Raj Pokkuluri for giving me the opportuni-

ty to learn about protein purification during my summer 2012 appointment. I would also like to

thank my program advisors at Northern Illinois University for providing me with the LOR’s that

are required to be accepted into the program. And finally my loving wife, whose support and

patients has helped to make it possible for me to work in one of the finest laboratories in the

world.