Selecting an Appropriate Method for Expressing a Recombinant Protein (1)
E. coli is normally inefficient in promoting the correct formation of disulfide bonds when recombinant proteins are expressed in the cytoplasm; normally disulfide bond formation occurs only in the periplasm where it is catalyzed by the Dsb system. Consequently, if disulfide bond formation is needed, the recombinant protein can be directed to the periplasm via a cleavable signal peptide.
P. pastoris is a methyltropic yeast, and can use methanol as its only carbon source. The growth of P. pastoris in methanol-containing medium results in the dramatic transcriptional induction of the genes for alcohol oxidase (AOX) and dihydroxyacetone synthase. After induction, these proteins comprise up to 30% of the P. pastoris biomass.
Stably transfected Chinese hamster ovary (CHO) cells have been reported to express recom- binant antibodies up to a level of a few grams per liter. HEK293 cells can be transiently transfected with a high efficiency (>80%) using certain cationic lipids, calcium phosphate, or polyethyleneimine as transfection reagents. The standard method for stable CHO expression involves transfecting dihydrofolate reductase (DHFR)-deficient CHO cells with a DHFR selection cassette along with an expression cassette containing the gene of interest. The entire selection and screening process takes at least 2–3 months, making this the major drawback of the CHO method. Mammalian cells contain the most superior folding and disulfide bond formation when compared to other expression hosts.
Expressing a eukaryotic protein containing several codons that are rare in E. coli can be inefficient when the pool of corresponding tRNAs is limiting. The shortage of tRNAs can lead to frameshifts in translation, misincorporation of amino acids, or premature termination of translation. For proteins between 10 and 50 kDa and containing few disulfide bonds, E. coli is a good option for soluble protein expression. Successful expression of proteins smaller than 10 kDa, with few or no disulfide bonds, has been achieved through fusion with soluble tags and expression in E. coli.
Normally, when generating recombinant proteins for use as immunizing antigens, any of the expression hosts can be used for this application. With glycoproteins it is not always clear whether the presence of glycans will alter the immunogenicity of the recombinant antigen.