The goal of this proposal is to develop a peptide cleavage system which will improve the efficiency and specificity of cleaving affinity tag peptides from recombinant proteins. The purpose is to simplify the purification of recombinant proteins from either prokaryotic or eukaryotic expression systems and ensure no additional terminal amino acids will remain on the final product. Although fusion tag peptides and peptidases are commercially available, the efficiency and specificity of cleavage is not optimal and often results in low yields of the desired protein product. Development of an optimized cleavage system will impact research labs as well as therapeutics and production processes by increasing the efficiency of studying new proteins and reducing the cost of protein production. A selectable M13 filamentous phage display system will be used to identify optimal combinations of cleavage sites and peptidases. Two approaches will be pursued: one targeting the peptide cleavage site, and the other targeting the enzymatic domain of the peptidase.
Several commercial applications will result from this work. The vectors constructed to perform the screening experiment will be marketable cloning vectors. The sequences identified through the research will be inserted into optimized protein expression vector. Modified peptidases will be marketed if they prove to be superior to naturally found enzymes.
