281740 A Rapid Approach to the Development of Affinity Reagents Using Yeast Surface Display

Wednesday, October 31, 2012: 8:30 AM
Westmoreland West (Westin )
James A. Van Deventer, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, Sachdev Sidhu, Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada and K. Dane Wittrup, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

Affinity reagents form a key part of many biological assays, diagnostic tests, and therapeutics, but their generation can require considerable time, effort, and expense. In this work, we present an approach to the rapid isolation of affinity reagents from a naive, synthetic antibody library using yeast surface display. A billion-member single chain variable fragment (scFv) library has been constructed from a minimalist library design based on a common framework region and restricted diversity in the complementarity determining regions. This library enables rapid, yeast-based isolation of affinity reagents from the naive library in approximately two weeks using a combination of bead- and flow cytometry-based sorting. Initial characterizations suggest that isolated clones exhibit low double-digit nanomolar affinities toward target antigens, comparable to many commercially available antibodies. Furthermore, the isolated scFv clones can be converted to a soluble antibody-like structure using a single ligation-free cloning step, enabling rapid validation in standard biological assays. Current efforts are focused on cloning-free conversion between displayed and secreted proteins and flow cytometry-free approaches to isolating individual binding proteins. We expect this approach to be useful to the scientific community for the development of custom affinity reagents for use in immunofluorescence microscopy, western blots, immunoprecipitations, flow cytometry, and other standard biological assays.

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