Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

ABSTRACT: While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by muta

SUBMITTER: Pershad K 

PROVIDER: S-EPMC3488158 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature