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 sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), We demonstrate that this information can be used to direct selective 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 were introduced into the 43-52 (or Omega) loop of staphylococcal nuclease, Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and K-m values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides, Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility, Cleavage of proteins by t-PA and u-PA is sequence selective, u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.