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Merck
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  • Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase.

Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase.

PloS one (2016-01-06)
Renhua Huang, Pete Fang, Zengping Hao, Brian K Kay
摘要

Affinity reagents of high affinity and specificity are very useful for studying the subcellular locations and quantities of individual proteins. To generate high-quality affinity reagents for human Lyn tyrosine kinase, a phage display library of fibronectin type III (FN3) monobodies was affinity selected with a recombinant form of the Lyn SH3 domain. While a highly specific monobody, TA8, was initially isolated, we chose to improve its affinity through directed evolution. A secondary library of 1.2 × 109 variants was constructed and screened by affinity selection, yielding three variants, two of which have affinities of ~ 40 nM, a 130-fold increase over the original TA8 monobody. One of the variants, 2H7, displayed high specificity to the Lyn SH3 domain, as shown by ELISA and probing arrays of 150 SH3 domains. Furthermore, the 2H7 monobody was able to pull down endogenous Lyn from a lysate of Burkitt's lymphoma cells, thereby demonstrating its utility as an affinity reagent for detecting Lyn in a complex biological mixture.

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Sigma-Aldrich
单克隆抗-FLAG® M2 小鼠抗, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
抗多组氨酸−小鼠单克隆甲状腺过氧化物酶抗体 小鼠抗, clone HIS-1, purified from hybridoma cell culture