Cell Free Protein Labeling with Molecular Partners

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 1R43GM067318-01
Agency Tracking Number: GM067318
Amount: $99,969.00
Phase: Phase I
Program: SBIR
Awards Year: 2003
Solicitation Year: N/A
Solicitation Topic Code: N/A
Solicitation Number: N/A
Small Business Information
HUBZone Owned: N
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 (617) 975-0685
Business Contact
Phone: (617) 975-0688
Research Institution
DESCRIPTION (provided by applicant): Cell free protein expression labeling provides a means for incorporation of fluorescent markers (fluorotags) and affinity tags (affytags) into proteins which can be used for a variety of biotechnological applications including proteomics and drug discovery. It relies on the attachment of a non-native amino acid to a specific tRNA using non-enzymatic chemical aminoacylation and the introduction of the misaminoacylated tRNA into a cell-free protein expression system. Such labels allow cell-free expressed proteins to be tracked, purified, isolated, conformation changes measured and interaction with other molecules detected. However, this approach is severely limited by restrictions imposed by the protein translation system on the structure of the amino acid side chains that can be incorporated into the protein. The proposed research will develop and evaluate a new approach, which circumvents labeling restrictions imposed by the protein translational apparatus. Molecular partners, small organic molecules, which exhibit highly selective and strong interaction between each other are utilized. By incorporating one of these partners into the protein and by using the other as a linker, versatile labeling and surface attachment of proteins can be achieved. Phase I will focus on the use of 1,3-phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA) derivatives as molecular partners. A novel class of affinity ligands, which form highly stable complexes with PDBA will be synthesized based on bis-salicylhydroxamic acid (bis-SHA). As opposed to currently used mono-SHA derivatives, these compounds are expected to have superior properties including complex stability and a functional group that enables easy conjugation with fluorotags, affytags and surfaces. A variety of applications of this new labeling technology will be developed during Phase II including utilization of mass spectrometry to analyze protein-protein interactions.

* information listed above is at the time of submission.

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