PEDS Advance Access published online on March 14, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl003
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1 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
* To whom correspondence should be addressed. Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein
Received July 15, 2005
Revised December 2, 2005
Accepted January 30, 2006
Article
Limitations of yeast surface display in engineering proteins of high thermostability
Sheldon Park 1,
Yao Xu 1,
Xiaoran Fu Stowell 2,
Feng Gai 3,
Jeffery G. Saven 3,
and
Eric T. Boder 3 *
2 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA; Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA; Laboratory for Research on the Structure of Matter, University of Pennsylvania, Philadelphia, PA 19104, USA
Eric T. Boder, E-mail: boder{at}seas.upenn.edu
Abstract 
3D were studied using yeast surface display. A library of 3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type 3D. Two other related sequences (3A and 3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.3D; helical protein; protein library; yeast surface display.
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