We propose to form a research collaboration in which we improve upon the screening and evaluation technologies currently used to generate affinity reagents. We intend to refine the discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. This Center will involve the laboratories of Drs. Brian Kay (University of Illinois at Chicago), Andreas Pluckthun (University of Zurich), and Michael Weiner (lllumina Corporation). We will focus on three types of recombinant affinity reagents: human single-chain Fragments of variable regions (scFv), Designed Ankyrin Repeat Proteins (DARPins), and fibronectin type III (FN3) monobodies. Dr. Aled Edwards (Structural Genomics Center) will supply us with 100 targets to which we will apply our technology improvements. We will also use synthetic peptides, corresponding to the N-termini and C-termini of a test set of proteins, as targets. Our research plan consists of three strategic goals. First, through molecular engineering we will generate versatile affinity reagents that can accomplish the many standard tasks of antibodies, as well as function as biosensors and inducible inhibitors of intracellular targets. Second, we plan to improve our current selection protocols by using emulsions to make them faster, smaller, cheaper, and better. Third, in addition to our own characterization, we will distribute affinity reagents to five beta-testers for their evaluation in biological applications and receive feedback on what additional properties should be engineered into the affinity reagents. Once we have prepared and vetted high quality affinity reagents, they will be made available through the DNA repository at Arizona State University and two reagent supply companies. The overarching goal of our research will be to optimize technologies that can be applied to the entire human proteome in a versatile, powerful, cost-effective, and high-throughput manner.

Public Health Relevance

There are -20,500 proteins encoded by the human genome, but there are very few tools available to quantify, purify, and visualize them in human cells and tissues. Thus, it is important to develop technologies that generate high quality affinity reagents in a cost-effective and high-throughput manner.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZRG1-BST-M (51))
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Sechi, Salvatore
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University of Illinois at Chicago
Schools of Arts and Sciences
United States
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Huang, Renhua; Fang, Pete; Hao, Zengping et al. (2016) Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase. PLoS One 11:e0145872
Venegas, Leon A; Pershad, Kritika; Bankole, Oluwadamilola et al. (2016) A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides. N Biotechnol 33:537-43
Huang, Renhua; Gorman, Kevin T; Vinci, Chris R et al. (2015) Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step. Int J Mol Sci 16:23587-603
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