Abstract
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 Ω) loop of staphyloooccal 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.
Original language | English (US) |
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Pages (from-to) | 4323-4328 |
Number of pages | 6 |
Journal | Journal of Biological Chemistry |
Volume | 273 |
Issue number | 8 |
DOIs | |
State | Published - Feb 20 1998 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology