Recombinant proteins are useful for studying biological processes and the structure of protein. Selecting an appropriate expression system is a crucial factor to produce correctly folded protein. Factors to consider when choosing an expression system include:
- Type of protein
- Number of disulfide bonds
- Type of post translational modifications
- Yield required
- Downstream Application
Post translational modifications are one of the most important factors to be considered when choosing an expression system. Post translational modifications are nothing but attaching a biochemical group such as acetate, phosphate, carbohydrate moieties and lipids to amino acid side chains that alter the biochemical and physical properties of a protein after translation. Many proteins undergo post translational modifications shortly after their translation and some after protein folding and some after localization. Most common protein post translational modifications are:
- Phosphorylation
- Glycosylation
- Methylation
- Ubiquitination
- S-Nitrosylation
- N-Acetylation
E. coli expression system, Yeast expression system, Insect cell expression system, and mammalian expression system are compared here with the context of post translational modifications. Each expression system has its own capabilities to carry out these post translational modifications. Expression of some proteins is toxic to one expression system while it is not in the other system. E. coli expression system is usually do not offer post translational modifications except few E. coli variants like Rosetta. E. coli system is used to express prokaryotic proteins and some eukaryotic proteins which do not require post translational modifications. In contrast yeast, insect and mammalian expression systems promote proper protein folding and post translational modifications.
E. coli expression system doesn’t support N- and O- linked glycosylation, hydroxylation, amidation, sulfation or palmitation. Yeast expression system is more advanced than E. coli in that it generates disulfide bonds efficiently. Yeast support both N- and O-linked glycosylations, but the glycan structures vary from that added by insect and mammalian systems. However recent advances in yeast systems offer glycan structures similar to mammalian cells. Insect cells are highly similar to mammalian cell expression system, helps in disulfide bond formation, glycosylation etc., But the old version of insect cells offer N-linked fucosylated pausimannose structures, recent advances lead to the generation of insect cells that offer complex N-linked glycans similar to mammalian cells. There are very few reports of insect cells offering O- linked glycosylation. However, mammalian expression system is superior over other expression systems in protein folding, disulfide bond formation and the post translational modifications like N-and O-linked glycosylation. Drawbacks of this system is low protein yield, time consuming (months for expression) and require expensive media, it can be chosen if the protein needs post translational modifications for its functionality.
For example,
- E. coli expression system: Some codons are not present in E.coli system which cause problems in protein translation called Rare codons or low usage codons. Recent modified E.coli cells contain these rare codons that support proper protein expression, folding, required post translational modifications to some extent. BL21 Codon plus cells contain tRNA with rare codons like AGA, AGG, AUA, CUA. Rosetta DE3 contain tRNA with Rare codons like AUA, AGG, AGA, CUA, CCC, GGA. Origami strains and Shuffle T7 cells support disulfide bond formation. C41 (DE3) and C43 (DE3) cells are effective in expressing toxic and membrane proteins from both prokaryotes and eukaryotes including viral proteins.
- Yeast cell expression system: Saccharomyces cerevisiae, Pichia pastoris are the two mostly used yeast for expressing protein mammalian like proteins with post translational modifications. Post translational modifications like N-terminal methionine removal and N-acetylation are conserved among yeast and mammalian cells, phosphorylation is the most common post translational modifications in yeasts, they can perform glycosylation of proteins similar to that of mammalian. Protease deficient strains like PichiaPink™ Strain 4 (ade2, prb1, pep4) are used to produce proteins with post translational modifications and to reduce protein degradation during When choosing yeast system for expression care should be taken while choosing the vectors and promoters as well. Glycolytic promoters (ADH1, GAP), phosphate regulated promoters (ADH2, CYC1), Galactose regulated promoters (GAL1, GAL7) are a few promoters used. Some proteins are toxic to cells when over expressed, so low copy plasmids have to be used (Yeast centromere plasmids YCp). High copy plasmids are used for higher yields of non-toxic proteins.
- Insect cell expression system: Insect cell expression system is gaining importance in biopharmaceutical industry after the approval of vaccines like cervical cancer vaccine Cervarix, Influenza vaccine FluBIok. Both Spodoptera fugiperda derived cell line, SF9 and Trichoplusia ni, BTI-Tn5B1-4 (High Five) derived cell lines are used to express intracellular and membrane proteins, but high protein yield is obtained with High five cells than SF9. Though both the cells offer post translational modifications, they differ to some extent from that of mammalian. Baculovirus based vectors like pBAC, pFastBac can be used.
- Mammalian cell expression system: Mammalian expression system is an excellent system that offer proper protein folding, disulfide bond formation and post translational modifications. It is very much of interest to researchers developing biologics. But the yields are very less compared to E.coli expression system and yeast expression system. HEK 293, CHO cells strains used previously, but the protein yield is less (20-200mg/L), new variants are developed like Expi293 and ExpiCHo systems that are used for high protein yield (1-3g/ L). One can choose from a range of vectors like SV40, pCMV, pSV, vaccinia, retroviral vectors depending on type of protein and whether transient or stable protein expression is required.
References:
- Jenkins N, Parekh RB, James Getting the glycosylation right: implications for the biotechnology industry. Nature Biotechnology, Aug :14 (8) : 975-81.
- Alexander A. Tokmakov Atsushi Kurotani, Tetsuo Takagi, Mitsutoshi Toyama, Mikako Shirouzu, Yasuo Fukami and Shigeyuki Yokoyama . Multiple Post- translational Modifications Affect Heterologous Protein Synthesis. The Journal of Biological Chemistry, June:12 287, 27106-27116.