RAPID IDENTIFICATION OF HIGHLY-ACTIVE AND SELECTIVE SUBSTRATES FOR STROMELYSIN AND MATRILYSIN USING BACTERIOPHAGE PEPTIDE DISPLAY LIBRARIES

The discovery of useful peptide substrates for proteases that recogniz e many amino acids in their active sites is often a slow process due t o the lack of initial substrate data and the expense of analyzing larg e numbers of peptide substrate. To overcome these obstacles, we have m ade use of bacteriophage peptide display libraries. We prepared a rand om hexamer library in the fd-derived vector fAFF-1 and included a ''te ther'' sequence that could be recognized by monoclonal antibodies. We chose the matrix metalloproteinases stromelysin and matrilysin as the targets for our studies, as they are known to require at least 6 amino acids in a peptide substrate for cleavage. The phage library was trea ted in solution with protease and cleaved phage separated from uncleav ed phage using a mixture of tether-binding monoclonal antibodies and P rotein A-bearing cells followed by precipitation. Clones were screened by the use of a rapid screening assay that identified phage encoding peptide sequences susceptible to cleavage by the enzymes. The nucleoti de sequence of the random hexamer region of 43 such clones was determi ned for stromelysin and 23 for matrilysin. Synthetic peptides were pre pared whose sequences were based on some of the positive clones, as we ll as consensus sequences built from the positive clones. Many of the peptides have k(cat)/K-M values as good or better than those of previo usly reported substrates, and in fact, we were able to produce stromel ysin and matrilysin substrates that are both the most active and small est reported to date. In addition, the phage data predicted selectivit y in the P-2 and P'(1) positions of the two enzymes that were supporte d by the kinetic analysis of the peptides. This work demonstrates that the phage selection techniques enable the rapid identification of hig hly active and selective protease substrates without making any a prio ri assumptions about the specificity or the ''physiological substrate' ' of the protease under study.