Eukaryotic Expression Systems

8/13/2019 Eukaryotic Expression Systems

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Eukaryotic expression

systems


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Some problems of expression in E. coli

Toxic proteins

Use a tightly regulated sytem

Induce late

Secretory proteins

Special strains are available

Expression of proteins with E. colirarecodons

Use special strains


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Rare codon-containing gene expression

Depletes the endogenous pool ofcorresponding tRNAs.

The deficit of tRNA molecules disrupts

translation,Truncated or no protein expression

Frameshifts, codon skipping, mis-

incorporations.Protein expression is slowed or aborted

mRNA is degraded.


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To resolve codon bias problemAlter the codon specifications of the

heterologous gene by site-directed

mutagenesisUse special strains expressing the

rare codons

Clone into eukaryotic expressionsystems.


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Protein aggregations caused by improperfolding due to

Attempt to express truncated protein

Protein with disulphide bonds (S-S aredifficult to form in cytoplasm of E. coli)

Try strains that are trxB, gsh, gor mutants,

Or trxB gor double mutants


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Deficiency in the chaperon machinery

Native state Folding is o ften catalysed by

ei ther molecular chaperones or folding

catalysts such as disu l f ide oxidases or

disul f ide isomerases and prol ineisomerases. Co-over-exp ression of those

can be helpful .

Proteolysis during or after folding or aftercell disruption.

Use the degP and ompT mutant strains

Add protease inhibitors to cell lysate


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It is difficult to express proteins for which Post-

translational Modifications are a must

Glycosylation does not work in E. coli

In vivo functional studies not possible inE. coli

The eukaryotic expression system attempts to

address some limitations of protein expression in

E. coli


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Eukaryotic expression systems

Of increasing popularityDue to their capability to perform many post-

translational modifications.

Main expression systems include

Yeast expression system,

Insect cell expression system and

Mammalian cell expression system

Xenopusoocytes

Plant expression e.g. Vaccine production in

beans, potatoes, maize and tobacco.


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Yeast expression system

The methylotropic yeast Pich ia pastor is

Usually utilizes alcohol oxidase

promoter to drive the expression of

foreign gene.

Recently, a continuous fermentation

has been developed in Pich ia pasto r is

with the glyceraldehyde-3-phosphate

dehydrogenase (GAP) promoter.


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Pichia pastoris

Single-celled, easy to manipulate and culture.

An eukaryote capable post-translational modifications

Proteolytic processing,

Folding,

Disulfide bond formation and Glycosylation.

Thus, biologically active proteins produced (rather than

inactive inclusion bodies in E. coli)

The P. pastoris system is faster, easier, and less

expensive to use than higher eukaryotes (insect and

mammalian cell systems) and

Higher expression levels achievable.


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Pichia pastoris

Vectors with Alcohol oxidase(AOX1) promoter

Expression of theAOX1 gene is tightly

regulated and

induced with methanol

Soluble protein yields of 5% in shake-flask

cultures, or

30% in fermenter cultures.


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Pichia pastoris

Vectors with GAP promoter

Glyceraldehyde-3-phosphate dehydrogenase

gene.

For expression of products that are non

toxic to P. pastoris.

Does not involve the use of methanol,

which may be problematic in some instances


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Pichia pastoris

Protein can be expressedintracellularly or

secreted into the medium.

P. pastoris secretes only low levels ofendogenous proteins, its culture medium

contains no added proteins,

Secreted heterologous protein comprisesthe vast majority of the total protein in the

medium.


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Pichia pastoris

Thus, secretion serves as a major firststep in purification, separating the foreign

protein from the bulk of cellular proteins

The secretion signal sequence

from the S. cerevisiae factor pre-pro

peptide

P. pastoris acid phosphatase gene(PHO1)


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The insect cell expression system

Is Baculovirus-mediated

Considered to be safe, powerful, but

cell-lytic.

The Baculovirus-S2 system uses the

popular and genetically well

understood Drosphila S2 cells

These do not appear to be lysed after

infection.


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The baculovirus is

For over-expression of a protein in insect

cells or whole insect larvae.Very high levels of expression

The advantages over E. coli are the

proper post translational modificationsGlycosylation (probably not exactly like

mammalian glycosylation).

Phosphorylation,Myristolation

Palmitolation.


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The baculovirus system

requires homologous recombination inside

transfected insect cells.

Linearized baculovirus with part of an essential

gene missing

A transfer vector carrying the needed missing

pieceThe gene to be over-expressed is cloned into

the transfer vector,

Transfer vector has strong polyhedrin promoter

that normally controls formation of the major

baculovirus protein.

so the cell recombines them both to create a

complete baculovirus


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pBlueBac4.5 co-expresses -galactosidase when co-

transfected with linearlised viral genome

Part of lacZ is on the linearized genome, and an overlapping

part of it is in the transfer vector

Recombinant plaques that are blue. (Ease of screening and

selecting recombinant plaques) .

e.g. of a carrier vector

B l i i t


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Baculovirus expression system

Can splice introns,

But the results are better if a cDNA is used.

Possible modification on protein to be expressed

His tag with EK cleavage site

If secreted protein is desired

A honey bee mellitin signal sequence can befused in frame with your gene.

The product will then be targeted to the ER for

the secretion pathway.The signal sequence will be cleaved off in the

processing for secretion,

leaving a wild type native protein.


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Other systems

I d ibl M lli i t


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Inducible Mammallian expression system


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Expression in Xenopus oocytes

Used to produce proteins for specificsensitive bioassays.

The function of mutant ion channels made

in very small quantities can be measuredby patch clamp methods.

The oocyte system is not made for large

scale protein production, but it is suitable formore specialized purposes.


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Expression in Xenopus oocytes

Two approaches:

Inject DNA into the egg nucleus and get

transcription to occur,

Inject RNA into the cytoplasm and get

translation.

The injection is done with a

micromanipulator and a micro-pipette


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2 routes of expression inxenopusoocytes


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How do the various systems compare?


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Large scale protein expression

Makes use of fermentersTransformed microorganisims cultured in

thousands of litres of growth medium

Under controlled parameters (O2,temp )Cetrifugation to recover the products

French press to dirupt bacteria

Or secretory products recovered fromsupernatants

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Eukaryotic Expression Systems