A general strategy for the evolution of bond-forming enzymes using yeast display.

Proc Natl Acad Sci U S A
Authors
Keywords
Abstract

The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

Year of Publication
2011
Journal
Proc Natl Acad Sci U S A
Volume
108
Issue
28
Pages
11399-404
Date Published
2011 Jul 12
ISSN
1091-6490
DOI
10.1073/pnas.1101046108
PubMed ID
21697512
PubMed Central ID
PMC3136257
Links
Grant list
R01 GM065400 / GM / NIGMS NIH HHS / United States
Howard Hughes Medical Institute / United States