Cell Free Protein Labeling with Molecular Partners

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.