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Directed Evolution of Protein Thermal Stability Using Yeast Surface Display

  • Michael W. Traxlmayr
  • Eric V. Shusta
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

Abstract

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.

Key words

Thermal stability Conformationally specific ligand Thermal denaturation Yeast surface display Protein engineering Directed evolution 

Notes

Acknowledgments

This work was supported by the Austrian Science Fund (FWF; Erwin-Schrödinger-Fellowship J3496-N28) and the National Science Foundation USA (CBET-1403350).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of ChemistryBOKU-University of Natural Resources and Life SciencesViennaAustria
  2. 2.Department of Chemical and Biological EngineeringUniversity of Wisconsin-MadisonMadisonUSA

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