| Protein kinases, known for phosphorylating their protein substrates to relay signaling events in cells, contain 518 members and encompass ∼2% of human genes. One subgroup of the kinase family, Src family kinases (SFKs), including Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, and Yes, have been studied for their roles in cell proliferation, migration, differentiation and survival. Because SFKs members have a similar overall structure with highly conserved sequences, currently available antibodies fail to distinguish the active form of one kinase from other SFKs. As SFKs often phosphorylate substrates with similar amino acid sequence, current biosensors fail to discriminate activation of one kinase from other SFKs.;To generate affinity reagents that can be used to study the activation of individual member of SFKs, I used phage display technology for directed evolution of FN3 monobodies. To increase the efficiency of constructing a phage library of FN3 monobodies with Kunkel mutagenesis, I made three modifications to the previously published protocol. First, I incubated bacterial cells at 25°C instead of 37°C to achieve a 2- to 7-fold increase in the yield of the single-stranded DNA template. Second, with the introduction of restriction endonuclease sites into the diversified loops of the FN3 coding region, phage libraries with diversity up to 1010 and 99-100% recombinant were constructed. Finally, I designed a digestion- and ligation-free method for constructing secondary libraries, using DNA fragments amplified by error-prone and asymetric PCR as primer for conducting Kunkel mutagenesis. To demonstrate the efficiency of this improved method, I constructed two secondary libraries based on a FN3 monobody that bound to the active form of Pak1 kinase. Screening one of the mutagenic libraries isolated three variants that bound 2- to 4-fold tighter than the original clone.;For generating affinity reagents to one member of the SFKs, Fyn tyrosine kinase, a phage-display library was screened for monobodies binding to the Fyn SH3 domain. The affinity selection identified three monobodies that bound selectively to the Fyn SH3 domain. One of the isolates, G9, bound exclusively to the Fyn SH3 domain out of 150 SH3 domains that I tested and had a dissociation constant (KD) of 166 ± 6 nM. Interestingly, while the G9 monobody lacks proline in its randomized loops, it bound at the same site on the SH3 domain as proline-rich ligands. The G9 monobody could be used to pull-down active recombinant Fyn kinase in vitro, demonstrating its potential as a highly selective probe for detecting active cellular Fyn kinase.;To generate affinity reagents for another SFKs member, Lyn tyrosine kinase, another phage library was screened. Two isolates, TA1 and TA8, selectively bound to the Lyn SH3 domain out of 150 SH3 domains. Both TA1 and TA8, in the absence of a canonical PxxP motif in their binding loops, competed with a PxxP proline-rich peptide, Tip, for binding to Lyn SH3 domain. Due to the weak affinity of TA8 for Lyn SH3 (KD= ∼5 µM), I used affinity maturation techniques to identify variants that bound > 8-fold tighter than the original TA8 clone. As a proof-of-concept experiment, one monobody was converted into a sensor that increased fluorescence upon binding to the Lyn SH3 domain in solution. |
