title: 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 mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display.
authors.affiliation: Department of Biological Sciences, Laboratory for Molecular Biology (M/C 567), University of Illinois at Chicago, Molecular Biology Research Building, Chicago, IL 60607, USA. kpersh2@uic.edu
authors.forename: Kritika
authors.initials: K
authors.lastname: Pershad
authors.forename: Karolina
authors.initials: K
authors.lastname: Wypisniak
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D051858:Forkhead Transcription Factors / Q000737:chemistry / Q000235:genetics*; D007202:Indicators and Reagents; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
chemical_list: D051858:Forkhead Transcription Factors; D007202:Indicators and Reagents