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Probing a Protein-Protein Interaction by in vitro Evolution

George Thom, Alexis C. Cockroft, Andrew G. Buchanan, Cathy Joberty Candotti, E. Suzanne Cohen, David Lowne, Phill Monk, Celia P. Shorrock-Hart, Lutz Jermutus and Ralph R. Minter
Proceedings of the National Academy of Sciences of the United States of America
Vol. 103, No. 20 (May 16, 2006), pp. 7619-7624
Stable URL: http://www.jstor.org/stable/30049306
Page Count: 6
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Probing a Protein-Protein Interaction by in vitro Evolution
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Abstract

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an $IC_{50}$ of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

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