Yeasts yeast display

Gai, S.A. and Wittrup, K.D. (2007) Yeast surface display for protein engineering and characterization. Current Opinion in Structural Biology, 17, 467 473. 

Boder, E. T. and Wittrip. K. D. (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotech. 15,553-558. 

Kieke, M. C., Shusta, E. V., Boder, E. T., Teyton, L., and Wittrup, K. D. (1999). Selection of functional T cell receptor mutants from a yeast surface-display library. Proc. Natl. Acad. Sd. USA 96, 5651-5656. 

Examples of such protein-protein interaction selection systems are phage display (Smith, 1985 Winter et al., 1994), display on other viruses (Kasahara et al., 1994), bacterial surface display (Georgiou et al., 1993 Daugherty et al., 1999), yeast display (Kieke et al., 1997 Boder and Wittrup, 1997), the yeast two hybrid system (Fields and Song, 1989 Chein et al., 1991), and protein-fragment complementation assays (Pelletier et al., 1998). These methods all contain a necessary in vivo step, which has a number of disadvantages that will be discussed in the following sections. 

Yeast display has been proposed recently as an alternative way to display mammalian proteins. Because some eukaryotic proteins expressed in E. coli are not in the soluble form, they cannot be incorporated into phage particles. Furthermore, phage display may sometimes select for reduced host toxicity or higher infectivity rather than increased affinity. Yeast display should alleviate expression biases present in E. coli and, as bacteria, have the advantage of high expression levels of displayed fusion polypeptides and selection through flow cytometric cell sorting. 

Finally, the use of yeast display to affinity-mature antibodies has also been reported [69]. A randomly mutated scFv library was displayed on the surface of yeast and selected using flow cytometry however, only a modest decrease in the koff rate was reported. 

Lipovsek D, Antipov E, Armstrong KA et al (2007) Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display. Chem Biol 14 1176-1185... 

The difficulty of separation is highly dependent on peak spreading, as shown in Fig. 6.5. It is therefore critical to minimize the peak width as far as possible. This would be difficult for cell display methods if only single color fluorescent labeling were used, because the primary source of variability is biological. Flow cytometry instrumentation point spread functions generally contribute below 2 % to the overall coefficient of variance (CV = standard deviation/mean), but typical overall CVs for yeast display are approximately 50 - 100 % for the logarithmic fluorescence intensity. 

Fortunately, two-color fluorescent labeling can be used to normalize ligand binding levels vs. expression levels, as shown in Fig. 6.6. The net effect of such normalization is to dramatically reduce the effective C V, by implicitly calculating the ratio of bound ligand levels to the level of cell-surface protein display. An example of the discrimination achievable by such normalization is illustrated for the actual case of an enriched mutant population from a yeast display affinity maturation project, in Fig. 6.7. 

Thanks to Jennifer Cochran, Katarina Midelfort, and Andrew Girvin for critical comments on this chapter. Thanks to Christilyn Graff for providing the data for Figs 6.6 and 6.7 prior to publication. The yeast display work reviewed here was supported by grants from the National Institutes of Health and the Whitaker Foundation. 

Miller CA 3rd. 1999. A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures. Toxicol. Appl. Pharmacol. 160 297-303... 

Yeast display has come a long way since proof of concept in 1993 to the successful expression of a human cDNA library in 2005 and the demonstration that small-molecule-binding proteins can be isolated in 2007. Considering the rapid progress over the last two years and the inherent advantages over in vitro and other in vivo methods, it is clear that yeast display will soon overcome the current limitations and possibly become the method of choice for isolation of natural product-binding proteins. 

Biosensor Detection Systems Engineering Stable, High-Affinity Bioreceptors by Yeast Surface Display... 

Key words Yeast surface display, Directed evolution, Affinity maturation, Thermal stability. 

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