Yeast surface 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. 

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... 

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

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

In this chapter, we describe detailed protocols for expression, mutation, and selection of optimal bioreceptor proteins by yeast surface display. Through this process, bioreceptor mutants can be engineered with greater stability for storage and regeneration conditions, as well as higher affinities for the detection of very low ligand concentrations. 

Gene Construction of Yeast Surface Displayed Bioreceptor... 

This section will describe a robust method for engineering a binding protein of interest using yeast surface display technology. Similar methods for gene engineering via yeast display have been reported (9, 31, 32), and slight variations may be used for particular applications to attain optimal results. 

Yeast surface display gene construct diagram... 

Fig. 1. Yeast surface display elements (A) Plasmid map of the yeast surface display vector pCT302 with a single-chain T cell receptor (scTCR) cloned in as a fusion with the yeast mating agglutinin protein Aga-2. Map was prepared with the help of PlasMapper (33). The open reading frame containing Aga-2 and the scTCR is expanded to show the important elements and restriction sites. (B) Diagram of a bioreceptor displayed on the surface of yeast fused to Aga-2. A detection scheme involving biotinylated ligand bound to the bioreceptor and fluorescent streptavidin may be used to analyze bioreceptor libraries on the surface of yeast.

Increased thermal stability of proteins used for biosensors is desirable to allow for robust devices that can withstand a variety of storage, assay, and regeneration conditions. In addition, some evidence suggests that starting from the most stable version of a bioreceptor by yeast surface display will aid in later affinity maturation efforts (38-40). In addition, thermally stable mutants can enable higher expression levels as soluble proteins from yeast or E. coli (19, 20, 38). Thermal stability selection rounds have been carried out on many of the proteins that were later mutated for high affinity (17,19-21, 38 2). 

Boder, E. T., Bill, J. R., Nields, A. W., Mar-rack, P. C. and Kappler, J. W. (2005) Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide. Biotechnol Bioeng 92, 485—491... 

Lipovsek, D., Lippow, S. M., Hackel, B. J., Gregson, M. W., Cheng, P., Kapila, A. and Wittrup, K D. (2007) Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display molecular convergence with single-domain camelid and shark antibodies. J Mol Biol 368, 1024-1041... 

Swers, J. S., Kellogg, B. A., and Wittrup, K. D. (2004) Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display. Nucleic Acids Res 32, e36... 

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