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An enhanced approach for engineering thermally stable proteins using yeast display.

ABSTRACT: 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 70°C. 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.

SUBMITTER: Pavoor TV

PROVIDER: S-EPMC3449400 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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An enhanced approach for engineering thermally stable proteins using yeast display.

Pavoor Tej V TV Wheasler Jean A JA Kamat Viraj V Shusta Eric V EV

Protein engineering, design & selection : PEDS 20120705 10

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 ...[more]

PMID: 22767886

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