International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 3.1, pp. 71-72
Section 3.1.4.2. Yeast
a
National Cancer Institute, Frederick Cancer R&D Center, Frederick, MD 21702-1201, USA, and bCenter for Advanced Biotechnology and Medicine, Howard Hughes Medical Institute and University of Medicine and Dentistry of New Jersey – Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854–5627, USA |
Yeasts are simple eukaryotic cells. Considerable effort has been expended in studying brewers' yeast, Saccharomyces cerevisiae, and in developing plasmid systems and expression vectors that can be used in this organism. Recently, methylotrophic yeasts, most notably Pichia pastoris, have been developed as alternative systems that offer several advantages over S. cerevisiae. Although yeast expression systems are reasonably robust, the expertise required to use these systems effectively is not as widespread as the corresponding expertise for the manipulation of E. coli strains. Nor are the tools, media and reagents necessary to grow yeast and select for the presence of expression plasmids as broadly available as those used for E. coli systems. However, the increasing commercial availability of complete kits (such as Pichia expression systems from Invitrogen) is making yeast systems more accessible.
While yeast systems do offer some advantages relative to E. coli, these advantages are, in general, modest. One primary advantage, the ability to produce large amounts of biomass using simple, inexpensive culture media, is probably more important for industrial-scale protein expression than for most laboratory applications, even those involving crystallography, which requires more protein than most simple biochemical experiments. Yeast systems do not, in general, offer solutions to some of the most difficult problems encountered when trying to express recombinant proteins in E. coli. Specifically, the problem of mimicking the post-translational modifications found in higher eukaryotes (particularly glycosylation), which has not been solved for E. coli, has not been solved in yeast either. None of the available systems recapitulates the post-translational modifications found in higher eukaryotes. Additionally, yeast systems introduce some new problems not seen with E. coli expression systems, specifically genetic instability and hyperglycosylation, both of which are more problematic in S. cerevisiae than in Pichia.
Yeast systems are perhaps most valued for high-level production of secreted proteins. For some naturally secreted proteins, passage through the secretory pathway is necessary for proteolytic maturation, glycosylation and/or disulfide bond formation and is essential for proper folding or function. But secretion is complex, and numerous factors, such as the signal sequence, gene copy number and host strain, can be critical for high-level expression. Secretion can significantly simplify purification, since secreted recombinant proteins can constitute as much as 80% of the protein in the culture medium. However, degradation of secreted proteins can be a major problem. In some instances, proteolysis has been minimized by alteration of the pH of the culture medium, by addition of amino acids and peptides, and by use of protease-deficient strains (Cregg et al., 1993).
The rules for expression of proteins in yeast are not the same as those used either in E. coli or in higher eukaryotes. In yeast, as in E. coli, cDNA sequences from a higher eukaryote must be tailored for high-level expression, following rules that are fairly well understood. Yeast grows at 25–30 °C and has a slower growth rate than E. coli (under typical growth conditions, yeast has a doubling time of approximately 90 min, compared to 30 min for E. coli). Transformation of yeast can be achieved using competent cells, sphaeroplasts or electroporation, but by any technique it is less efficient than the transformation of E. coli. For these reasons, most yeast plasmids are designed to replicate both in E. coli and yeast; the DNA manipulations are done using an E. coli host, and the completed expression plasmid is introduced into yeast as the final step in the process.
Most expression vectors in S. cerevisiae are based on the yeast plasmid (Beggs, 1978; Broach, 1983) that is maintained as an episome, present at approximately 100 copies per cell. Plasmid instability can result in loss of expression during production, and integrating vectors have been developed that provide greater stability, albeit with levels of expression that are, in general, lower than the plasmid systems. Both constitutive and tightly regulated inducible expression systems have been developed using a variety of promoters. The most widely used systems involve galactose-regulated promoters, such as GAL1, which are capable of rapid and high-level induction. An extensive review of recombinant gene expression in yeast (Romanos et al., 1992) is highly recommended as a resource for anyone seriously contemplating the expression of recombinant proteins in S. cerevisiae.
In terms of high-level expression, the Pichia system may ultimately prove to be more useful than S. cerevisiae (for reviews see Cregg et al., 1993; Romanos, 1995; Hollenberg & Gellissen, 1997). There is considerable interest in developing the Pichia system for the expression of recombinant proteins, especially for industrial applications, and there has been sufficient progress made to support the publication of a useful monograph for specific techniques (Higgins & Cregg, 1998). Pichia offer several advantages over S. cerevisiae. Intracellular protein expression can be extremely high in Pichia, reaching grams per litre of cell culture. Large amounts of secreted proteins can be produced using media that are almost protein-free, although the expression levels are not quite as high as for intracellular proteins. Pichia can be cultured to very high cell density with good genetic stability. Additionally, hyperglycosylation is less of a problem in Pichia, which typically have shorter outer-chain mannose units (less than 30 outer-chain residues) than S. cerevisiae (greater than 50 residues) (Grinna & Tschopp, 1989).
Methylotrophic yeasts, which are able to use methanol as their sole carbon source, contain regulated methanol enzymes that can be induced to give extremely high levels of expression. In Pichia expression systems, the gene that encodes alcohol oxidase (AOX1) is most commonly used for the expression of foreign genes, but constitutive promoters are also available. Heterologous genes are inserted into vectors and then integrated into the Pichia genome, either duplicating or replacing (transplacement) the target gene, depending on how the linearized vector is constructed. High-level expression relies on integration of multiple copies of the foreign gene and, since this varies significantly, screening colonies to obtain clones with the highest levels of expression is required. Culture conditions and induction protocols are critical for optimal expression. Since Pichia are readily oxygen-limited in shake flasks, growth in fermenters is required for high-level expression (approximately five- to tenfold greater than in shake flasks).
Numerous factors make yeast expression systems significantly less straightforward than those of E. coli. In addition to the considerations mentioned above, it should be noted that yeast cells are surrounded by a tough cell wall and are therefore notoriously difficult to break. This makes the problem of purification of intracellular protein from yeast that much more difficult. Given the many complexities of expression in yeast, it is usually better to begin with an E. coli expression system and move to yeast only if the results obtained with E. coli systems are unacceptable. If yeast is used as an expression system, careful attention should be paid to maintaining defined stocks of the expression strain and the corresponding expression plasmids. Despite the availability of comprehensive kits, if the researcher does not have considerable experience with yeast, the enlistment of an experienced colleague is recommended.
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