Biotechnlogy lecture 4

8/7/2019 Biotechnlogy lecture 4

1/34

Biotechnology PH2204Les Baillie, room 1.54

4 of 11


2/34

t

Isolate

bacteria

containing

targetDNA

Transformation

selection

Transfer to large scale

expression hostPropagate colonies of interest

Antibiotics


3/34


expression host

E.coliis a good host for genetic manipulation

Not necessarily best host for the expression of

recombinant protein particularly those

destined for use in human


4/34

PA Expression systems

B.anthracis Sterne

Salmonella typhimurium

Lactobacillus casei

Bacillus brevisB.subtilis WB600 (protease def)

E.coli

E.coli(optimised codon usage)

mg/l PA

Second Generation Vaccine- rPA


5/34

Host cells

Bacteria (E. coli) Prokaryote

Yeast

(S. cerevisiae, P. pastoris)- E

ukaryot

e

Mammalian cells (C127, CHO, DON, HEK, BHK, COS)

Insect cells (High-five, Sf 9, Sf 21)

Transgenic animals (plants) (sheep, goat, cow)


6/34

Characteristics E. coli Yeast Insect cellsMammalian

cells

Cell growth rapid (30 min) rapid (90 min) slow (18-24 h) slow (24 h)

Complexity of growth medium minimum minimum complex complex

Cost of growth medium low low high high

Expression level high low - high low - highlow -

moderate

Extracellular expressionsecretion to

periplasm

secretion to

medium

secretion to

medium

secretion to

medium

Posttranslational modifications

Protein foldingrefolding usually

required

refolding may

be requiredproper folding proper folding

N-linked glycosylation none high mannose simple, no sialicacid

complex

O-linked glycosylation no yes yes yes

Phosphorylation no yes yes yes

Acetylation no yes yes yes

Acylation no yes yes yes

gamma-Carboxylation no no no yes

Merits of various approaches


7/34

Prokaryotes versus Eukaryotes


8/34


9/34

Large scale production

1000ml

10,000ml


10/34

Regulation of protein expression

Constitutive expression- protein made all ofthe time

Requires lots of energy- affect growth rate

Inducible expression protein made when you addthe inducer

Reduces energy requirements

express the protein when you have achieved sufficient

cell mass i.e 100,000,000 cells rather than 10,000 Allows the production of proteins which are toxic to

the host


11/34

http://www.youtube.com/watch?v=oBwtxdI1zvk


12/34

Constitutive expression

P

Promoter

Protein Z Protein Y Protein A

> Y A

RNA pol

In order for transcription to take place, the enzyme that synthesizes RNA, known as RNA

polymerase, must attach to the DNA near a gene at a region known as the promoter.

Promoters contain specific DNA sequences and response elements which provide a binding site

for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase.

In bacteria, the promoter is recognized by RNA polymerase and an associated sigma factor,

which int

urn are oft

en brought

t

ot

he promot

er DNA by an act

ivat

or prot

ein bindingt

o it

s ownDNA binding site nearby.


13/34

Inducible promoter

Gene under the control of an inducible

promoter are only expressed in the presence

of specific signals

To achieve this they have additional genetic

control elements


14/34

lac I P O

Promoter

lac repressor

lac Z lac Y lac A

The lactose operon in E. coli

F-galactosidase permease acetylase

LACTOSE GLUCOSE + GALACTOSEF-galactosidase

Function of the lactose (lac) operon is to produce theenzymes which metabolize lactose to glucose and

galactose which are used to produce energy

Operator


15/34

When lactose is not present in the cell

lac repressor

lac I P lac Z lac Y lac A

the repressor tetramer binds to the operator

NO TRANSCRIPTION

RNA pol

RNA polymerase is blocked from the promoter


16/34

when lactose becomes available, it is taken up by the cell

allolactose (an intermediate in the hydrolysis of lactose) is produced

one molecule of allolactose binds to each of the repressor subunits

When Lactose is present

allolactose

lacI

P lac Z lac Y lac A

IPTG (isopropyl thiogalactoside) can also be used as an inducer


17/34

lac I P lac Z lac Y lac A

RNA pol

O

repressor (with bound allolactose) dissociates from the operator

negative control (repression) is alleviated, however...

RNA polymerase cannot form a stable complex with the promoter

NOTR

AN

SCRI

PTION


18/34

lac I P O lac Z lac Y lac A

Additional level of regulationin the absence of glucose cells synthesize cyclic AMP (cAMP)

cAMP1 serves as a positive regulator of catabolite repressed operons (lac operon)

cAMP binds the dimeric cAMP binding protein (CAP)2

binding of cAMP increases the affinity ofCAP for the promoter

binding ofCAP to the promoter facilitates the binding ofRNA polymerase

active CAP inactiveCAPcAMP

+

2 also termed catabolite activator protein

RNA pol

RNA pol

TRANSCRIPTION

F-galactosidase permease acetylase


19/34

Pharmaceuticals produced from E.coli

Recombinant protein Condition

Human growth hormone Dwarfism

Interferon a Cancers and viral disease

Interferon g Chronic granulomatous disease

Tissue plasminogen activator Acute myocardial infarction &Pulmonary embolism

Relaxin Facilitates childbirth

a-antitrypsin Treatment of emphysema

Human insulin Diabetes


20/34

Generation of insulin

pBR322

cDN

A A subunit

cDNA LacZ

Beta-galactosidase

Linked through

methionine

pBR322

cDN

A B subunit

cDNA LacZ

Beta-galactosidase

Linked through

methionine

Clone in E.coli

Isolate product

Treat with cyanogen

bromide to cleave atmethionine a.a link

Under oxidising conditions


21/34

Proteolytic processing Insulin, an example of a nonglycosylated protein

Processing of insulin (synthesized in the ER of pancreatic F-cells)

N

C

Preproinsulin

110 aa

cleavage of

signal peptide

by signal

peptidase

Signal peptide

C

S

I

S

S

I

S

N

C-chain

Cleavage by trypsin-like enzymes

releases the C-peptide

B-chain

A-chain

C

S

IS

S

IS

N

Insulin 51 aa

CN

Further trimming by a

carboxypeptidase B-like

enzyme removes two

basic residues from

each of the new ends

C-chainThe C-chain is

packaged in the secretory

vesicle and is secreted along with active insulin

C

S

I

S

S

I

S

N

Proinsulin 86aa

Disulfide bond

formation


22/34

Some Eukaryote proteins unstable in bacteria or lackbiological activity dues to e.g. incorrect folding or incorrectpost-translational modifications

Prokaryotes lack the ability to undertake the following post-

translational modifications Disulphide formation

Proteolytic cleavage

Glycosylation

Recombinant proteins expressed at high levels are stored asinclusion bodies

Prokaryotes harbour pyrogens ( and thus the final product

requires extensive purification)

Problems with Prokaryotic systems


23/34

Protein structure


24/34

Yeast a Eukaryote system

Saccharomyces cerevisiae Pichia pastoris - engineered strains to perform human

glycosylations of high homogeneity

Post-translational processes similar tomammalian, can perform N-glycosylations

Tend to add sugars side chains of high mannosecontent - affinity for mamnose receptors

Genetics well known

Rapid growth

Listed as generally recognised as safe GRASwith regulatory authorities

Do not produce pyogens


25/34


26/34

Glycosylation

The process is one of the principal co-translational and post-translational modification steps in thesynthesis of membrane andsecreted proteins

~ 50% of all eukaryotic proteins synthesized in the rough ER areglycosylated. It is an enzyme-directed site-specific process, as

opposed to the non-enzymatic chemical reaction of glycation.

Two types of glycosylation exist: N-linked glycosylation to the amidenitrogen of asparagines side chains and O-linked glycosylation tohydroxy oxygen of serine and threonine side chains.

O-linked (Ser, Thr linked) oligosaccharides (linked to hydroxylgroup)

N-linked (Asn linked) oligosaccharides (linked to amide group)


27/34

The most common modifications occur through

N- or O- glycosidic bonds

O-linkage: Ser, Thr (GalNAc) N-linkage: Asn (GlcNAc)

The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose,

GalNAc and GlcNAc.

Glucose (Glc)

N-acetylglucosamine (GlcNAc)

Mannose (Man)

Galactose (Gal)

N-acetylneuraminic acid (-ve) (sialic acid or NANA)

Fucose (Fuc)

N-acetylgalactosamine (GalNAc)


28/34

GlycosylationCurrently > 100 protein products approved in USA and Europe with 500 candidates

In clinical or preclinical development. 70% ofthese are glycoproteins

Glycosylated products : full length IgG, hormones, EPO and G/M-CSF. Interferons..

Non-glycosylated protein forms or incorrect glycosylation patterns can:

Alter PK

Molecular size, charge, uptake by scavenging receptors, e.g. asialoglycoprotein receptor inliver, or mannose receptors ofthe RES clearing glycoproteins with terminal mannose

E.g. EPO contains 3 N-linked glycosylation sites that in the natural protein would contribute40% ofthe final mass ofthe glycoprotein. A glycosylated forms display good in-vitroactivity but poor in-vivo activity because of shorter half-life. Eg, in rodents EPO PK half-lifefalls from 5 hrs to < 2 min for the desialylated EPO.

Immunogenicity

E.g. xylose an oligosaccharide from plants

Altered pharmacological activity

E.g. IgG heavy chain contains a single N-linked glycosylation site, i.e. 2 sites for each IgGmolecule.

Aglycosylated forms display significant loss of antibody associated cell mediatedcytotoxicity

Varied glycosylation patterns display varying degrees of activity.


29/34

Disulphide bond formation

A disulfide bond is a single covalent bond derived from the coupling of thiol groups.

Disulfide bonds areusually formed from the oxidation ofsulfhydryl (-SH) groups, as

depicted.

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues

The disulfide bond stabilizes the folded form of a protein in several ways:

1)It holds two portions ofthe protein together, biasing the protein towards the folded topology.

2)The disulfide bond may form the nucleus of a hydrophobic core ofthe folded protein, i.e., local

hydrophobic residues may condense around the disulfide bond and onto each other through

hydrophobic interac

tions.3)The disulfide bond link two segments ofthe protein chain,

In eukaryotic cells, disulfide bonds are generally formed in the lumen ofthe RER (rough endoplasmic

reticulum) but not in the cytosol. This is due to the oxidative environment ofthe ER and the reducing

environment ofthe cytosol Thus disulfide bonds are mostly found in secretory proteins, lysosomal

proteins, and the exoplasmic domains of membrane proteins.


30/34

Gerngross Nature Biotech 22, 1409-1414


31/34

Characteristics E. coli Yeast Insect cellsMammalian

cellsCell growth rapid (30 min) rapid (90 min) slow (18-24 h) slow (24 h)

Complexity of growth medium minimum minimum complex complex

Cost of growth medium low low high high

Expression level high low - high low - highlow -

moderate

Extracellular expressionsecretion to

periplasm

secretion to

medium

secretion to

medium

secretion to

medium

Posttranslational modifications

Protein foldingrefolding usually

required

refolding may

be requiredproper folding proper folding

N-linked glycosylation none high mannose simple, no sialicacid

complex

O-linked glycosylation no yes yes yes

Phosphorylation no yes yes yes

Acetylation no yes yes yes

Acylation no yes yes yes

gamma-Carboxylation no no no yes

Merits of various approaches


32/34

Cell culture based systems- CHO cells

Mammalian cell of choice for production of glycoproteins

Produce N-glycans similar to those found in humans

Disadvantage High cost,

Long developmenttime from gene cloning to cell line

Pathways similar but not same as humans

e.g. EPO in CHO contains N-acetylneuraminic acid (Sialic acid)and N-glycolyneuraminic acid

propagation of infectious agents, e.g. viruses, prions


33/34

Insect based cell culture


34/34

t

Isolate

bacteria

containing

targetDNA

Transformation

selection


expression hostPropagate colonies of interest

Antibiotics

Biotechnlogy lecture 4

Documents

Transcript of Biotechnlogy lecture 4