Eukaryotic expression systems

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. coli rare codons Use special strains

Some problems of expression in E. coli

Rare codon-containing gene expression Depletes the endogenous pool of corresponding tRNAs. The deficit of tRNA molecules disrupts translation, Truncated or no protein expression Frameshifts, codon skipping, misincorporations. Protein expression is slowed or aborted mRNA is degraded.

Some problems of expression in E. coli

To resolve codon bias problem Alter the codon specifications of the heterologous gene by site-directed mutagenesis Use special strains expressing the rare codons Clone into eukaryotic expression systems.

Some problems of expression in E. coli

Protein aggregations caused by improper folding due to Attempt to express truncated protein Protein with disulphide bonds (S-S are difficult to form in cytoplasm of E. coli) Try strains that are trxB, gsh, gor mutants, Or trxB gor double mutants

Some problems of expression in E. coli

Deficiency in the chaperon machinery Native state Folding is often catalysed by either molecular chaperones or folding catalysts such as disulfide oxidases or disulfide isomerases and proline isomerases. Co-over-expression of those can be helpful. Proteolysis during or after folding or after cell disruption. Use the degP and ompT mutant strains Add protease inhibitors to cell lysate

Some problems of expression in E. coli

It is difficult to express proteins for which Posttranslational Modifications are a must Glycosylation does not work in E. coli In vivo functional studies not possible in E. coli

The eukaryotic expression system attempts to address some limitations of protein expression in

E. coli

Eukaryotic expression systems

Of increasing popularity Due to their capability to perform many posttranslational modifications. Main expression systems include Yeast expression system, Insect cell expression system and Mammalian cell expression system Xenopus oocytes Plant expression e.g. Vaccine production in beans, potatoes, maize and tobacco.

Yeast expression system

The methylotropic yeast Pichia pastoris Usually utilizes alcohol oxidase promoter to drive the expression of foreign gene. Recently, a continuous fermentation has been developed in Pichia pastoris with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

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.

Pichia pastoris Vectors with Alcohol oxidase (AOX1) promoter

Expression of the AOX1 gene is tightly

regulated and
induced with methanol

Soluble protein yields of 5% in shake-flask

cultures, or

30% in fermenter cultures.

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

Pichia pastoris
Protein can be expressed intracellularly or secreted into the medium. P. pastoris secretes only low levels of endogenous proteins, its culture medium contains no added proteins, Secreted heterologous protein comprises the vast majority of the total protein in the medium.

Pichia pastoris
Thus, secretion serves as a major first step 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)

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.

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 modifications Glycosylation (probably not exactly like mammalian glycosylation). Phosphorylation, Myristolation Palmitolation.

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 piece The 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

e.g. of a carrier vector

pBlueBac4.5 co-expresses -galactosidase transfected with linearlised viral genome

when

co-

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) .

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 be fused 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.

Other systems

Inducible Mammallian expression system

Expression in Xenopus oocytes

Used to produce proteins for specific sensitive bioassays. The function of mutant ion channels made in very small quantities can be measured by patch clamp methods. The oocyte system is not made for large scale protein production, but it is suitable for more specialized purposes.

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

2 routes of expression in xenopus oocytes

How do the various systems compare?

Large scale protein expression

Makes use of fermenters Transformed 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 from supernatants