Directed Evolution of Protein Thermal Stability Using Yeast Surface Display.

Yeast surface display is a powerful protein engineering technology that has been used for many applications including engineering protein stability. Direct screening for improved thermal stability can be accomplished by heat shock of yeast displayed protein libraries. Thermally stable protein variants retain binding to conformationally specific ligands, and this binding event can be detected by flow cytometry, facilitating recovery of yeast clones displaying stabilized protein variants. In early efforts, the major limitation of this approach was the viability threshold of the yeast cells, precluding the application of significantly elevated heat shock temperatures (>50 °C) and therefore limited to the engineering of intrinsically unstable proteins. More recently, however, techniques for stability mutant gene recovery between sorting rounds have obviated the need for yeast growth amplification of improved mutant pools. The resultant methods allow significantly higher denaturation temperatures (up to 85 °C), thereby enabling the engineering of a broader range of protein substrates. In this chapter, a detailed protocol for this stability engineering approach is presented.