Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 25, Issue 10, Pages 625-630Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzs041
Keywords
protein engineering; thermal stability; yeast surface display
Funding
- National Institutes of Health [NS052649]
- National Science Foundation [CBET-0966014]
- University of Wisconsin Graduate School
- NSF Graduate Research Fellowship
- NIH Biotechnology Training Grant Fellowship [GM08349]
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [0966014] Funding Source: National Science Foundation
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Many biotechnology applications require the evolution of enhanced protein stability. Using polymerase chain reaction-based recovery of engineered clones during the screen enrichment phase, we describe a yeast display method capable of yielding engineered proteins having thermal stability that substantially exceeds the viability threshold of the yeast host. To this end, yeast-enhanced green fluorescent protein destabilized by dual-loop insertion was engineered to possess a substantially enhanced resistance to thermal denaturation at 70C. Stabilized proteins were secreted, purified and found to have three- to six-fold increased resistance to thermal denaturation. The validated method enables yeast display-based screens in previously inaccessible regions of the fitness landscape.
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