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Directed Evolution of the Forkhead-associated Domain to Generate Anti-Phosphospecific Reagents

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posted on 22.02.2015, 00:00 authored by Kritika Pershad
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 domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated 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 50ºC prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8ºC more thermal 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 seven pT peptides. These reagents are renewable and have high protein yields (~20-25 mg/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.

History

Advisor

Stone, David E.

Department

Biological Sciences

Degree Grantor

University of Illinois at Chicago

Degree Level

Doctoral

Committee Member

Kay, Brian K. Jin, Hua Orenic, Teresa V. Santarsiero, Bernard D.

Submitted date

2012-12

Language

en

Issue date

21/02/2013