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Mempro™ Protein Production in Yeast Cells System

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Creative Biolabs can provide high-quality Mempro™ membrane protein production services using yeast cells, the two species of yeast most widely used for recombinant membrane protein production are Pichia pastoris (P. pastoris) and Saccharomyces cerevisiae (S. cerevisiae).

Single-celled yeast is a consistently popular choice in the eukaryotic membrane protein production field. As microbes, they are quick, easy and inexpensive to culture; as eukaryotes they are able to post-translationally modify eukaryotic membrane proteins. Recent crystal structures of recombinant trans-membrane proteins produced in yeast include human aquaporin 2, chicken bestrophin-1, the human TRAAK channel, human leukotriene C4 synthase, an algal P-glycoprotein homologue and mouse P-glycoprotein using P. pastoris; the structures of the Arabidopsis thaliana NRT1.1 nitrate transporter, a fungal plant pathogen TMEM16 lipid scramblase and the yeast mitochondrial ADP/ATP carrier were solved using recombinant membrane protein production in S. cerevisiae (Figure 1).

Mempro™ Protein Production in Yeast Cells System Figure 1. Overview of recombinant trans-membrane proteins produced in yeast cells. (Methods, 2016)

Creative Biolabs develops some strategies for improving yields per cell of P. pastoris and S. cerevisiae, through optimizing the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional membrane protein. The significant advantages of S. cerevisiae are described as below: 1). Its genetics are better understood, and 2). It is supported by a more extensive literature than P. pastoris. While P. pastoris has the advantage of being able to grow to very high cell densities and therefore has the potential to produce large amounts of recombinant membrane protein for structural analysis. This yeast species has also been important in generating high-resolution GPCR crystal structures such as the adenosine A2A and the histamine H1 receptors. Typically, episomal plasmids are used for expression in S. cerevisiae, and the expression cassette is integrated into the genome of P. pastoris. Since the P. pastoris system depends upon very strong promoters, only a few copies of the gene are required to obtain sufficient levels of mRNA. In contrast, in S. cerevisiae, the promoter can be 10- to 100-fold weaker so the use of episomal plasmids with high copy numbers is advantageous. The strong S. cerevisiae promoter, PGAL1, is induced with galactose while PAOX1 (a very strong P. pastoris promoter) is induced with methanol. In choosing a strong promoter, the idea is that transcription should not be rate limiting. However, high mRNA synthesis rates may be countered by high rates of mRNA degradation. Thus, a suitable balance between mRNA and protein synthesis rates is desirable, one possibility might be a system based on slow, constitutive expression.

Creative Biolabs can also provide other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.

References:
J. Petschnigg, et al. (2011). Using yeast as a model to study membrane proteins. Curr. Opin. Nephrol Hypertens, 20(4): 425-432.
N. Bonander, et al. (2012). Optimising yeast as a host for recombinant protein production. Methods Mol. Biol., 866: 1-9.
S. J. Routledge, et al. (2016). The synthesis of recombinant membrane proteins in yeast for structural studies. Methods, 95: 26-37.
Z. Bawa, et al. (2011). Understanding the yeast host cell response to recombinant membrane protein production. Biochem. Soc. Trans., 39(3): 719-723.




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