DIRECTING SEQUENCE-SPECIFIC PROTEOLYSIS TO NEW TARGETS - THE INFLUENCE OF LOOP SIZE AND TARGETS SEQUENCE ON SELECTIVE PROTEOLYSIS BY TISSUE-TYPE PLASMINOGEN-ACTIVATOR

We have previously used substrate phage display to identify peptide se quences that are efficiently and selectively cleaved by tissue-type pl asminogen activator (t-PA) or urokinase-type plasminogen activator (u- PA), We demonstrate that this information can be used to direct select ive proteolysis to new protein targets, Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide we re introduced into the 43-52 (or Omega) loop of staphylococcal nucleas e, Both t-PA and u-PA hydrolyze the engineered proteins at the inserte d target sequences, and K-m values for protein cleavage were reduced u p to 200-fold relative to values for cleavage of analogous sequences w ithin 15 residue peptides, Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more str ongly than that of trypsin, suggesting that cleavage by t-PA is more d ependent on target site mobility, Cleavage of proteins by t-PA and u-P A is sequence selective, u-PA is 47-fold more active than t-PA for cle avage of a sequence known to be u-PA selective within small peptide su bstrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.