Protein-capture reagents are indispensable for delineating the molecular mechanisms of diseases, to detect and characterize cellular abnormalities, and to characterize biological effects of drugs. However, the current paucity of high-quality protein-capture reagents presents a major bottleneck in virtually all areas of biomedical sciences. The overarching goal of this project is to develop an innovative and powerful protein-capture technology with high levels of fidelity and predictability.
We aim to overcome a major limitation of currently available technologies where specificity and epitopes must be individually tested by laborious methods after generating protein-capture reagents. We introduce a new concept, "C-clamping", that enables to direct capture reagents ("C-clamps") exclusively to the C-terminal (6-8) residues of proteins with high fidelity and high affinity. C-clamps are in the form of robust recombinant binding proteins generated using state-of-the-art phage-display technologies. Virtually every protein has a unique C-terminal signature that can be recognized with high efficiency by C-clamps. The a priori knowledge of epitope location allows one to accurately predict the level of specificity by identifying potential cross-reactivity through database search and to implement strategies to eliminate off-targets. These attributes make C-clamps particularly suited as the core technology for generating a comprehensive set of protein capture reagents. Furthermore, C-clamping is ideally suited to detect proteolytic "neo-epitopes" generated by proteolysis, markers of biomedically important processes (e.g. apoptosis). Our proof-of-concept experiments have successfully demonstrated the feasibility and enormous potential of C-clamping. Proposed studies aim to establish C-clamping as a general technology by producing high-performance capture reagents for high-value targets including integral membrane proteins, splice variants, viral proteins and caspase neo-epitopes. C-clamping represents a paradigm shift in capture-reagent generation, and the establishment of C-clamping will make large contributions to the entire molecular biomedical sciences.

Public Health Relevance

Detecting and measuring the amounts of proteins are critical for understanding differences between normal and diseased states of cells and tissues. This project will establish a totally new approach to facile generation of detection reagents that are high performance, easy to produce and easy to made available to the research community. This innovative and powerful technology will fill a major void in the currently available molecular tools and will have a major impact on virtually all areas of molecular biomedical sciences, diagnisis and drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090324-05
Application #
8539025
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Smith, Ward
Project Start
2009-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$494,781
Indirect Cost
$174,746
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Yasui, Norihisa; Findlay, Greg M; Gish, Gerald D et al. (2014) Directed network wiring identifies a key protein interaction in embryonic stem cell differentiation. Mol Cell 54:1034-41
Lu, Min; Symersky, Jindrich; Radchenko, Martha et al. (2013) Structures of a Na+-coupled, substrate-bound MATE multidrug transporter. Proc Natl Acad Sci U S A 110:2099-104
Koide, Shohei; Huang, Jin (2013) Generation of high-performance binding proteins for peptide motifs by affinity clamping. Methods Enzymol 523:285-302
Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine et al. (2013) Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains. Proc Natl Acad Sci U S A 110:14924-9
Koide, Akiko; Wojcik, John; Gilbreth, Ryan N et al. (2012) Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold. J Mol Biol 415:393-405
Koide, Shohei; Koide, Akiko; Lipovšek, Daša (2012) Target-binding proteins based on the 10th human fibronectin type III domain (¹?Fn3). Methods Enzymol 503:135-56
Gilbreth, Ryan N; Truong, Khue; Madu, Ikenna et al. (2011) Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design. Proc Natl Acad Sci U S A 108:7751-6
Huang, Jin; Koide, Shohei (2010) Rational conversion of affinity reagents into label-free sensors for Peptide motifs by designed allostery. ACS Chem Biol 5:273-7
Huang, Jin; Nagy, Stanislav S; Koide, Akiko et al. (2009) A peptide tag system for facile purification and single-molecule immobilization. Biochemistry 48:11834-6