title: Enzymatic condensation of nonassociated peptide fragments using a molecular trap.
abstract: We have tested the feasibility of achieving protease-catalyzed condensation between nonassociating peptide fragments through mediation of a molecular trap. In this study, two subfragments of bovine pancreatic ribonuclease S-peptide, containing residues 1-10 and 11-15, were rejoined by clostripain catalysis to form the 1-15 peptide. The extent of this stereospecific condensation was enhanced by adding ribonuclease S-protein (residues 21-124), which acts as a trap in binding 1-15 but not 1-10 or 11-15 and which thus shifts the equilibrium to favor 1-15 formation. The resultant (1-15) X (21-124) noncovalent complex, defined as [des-16-20]ribonuclease S, was detected by the enzymatic activity characteristic of the naturally derived ribonuclease S complex. Reaction of 1 mM S-protein and 20 mM fragments leads to 80% of the ribonuclease activity expected from the amount of 21-124 present. This indicates that 4% of the fragments 1-10 and 11-15 were condensed, compared to a maximal condensation of 5% based on the amount of trap. The less than theoretical yield is due largely to slow proteolytic degradation of 21-124 to a form which is no longer able to bind the condensation product 1-15. Yields were increased to 15% by addition of further trap. The successful synthesis of 1-15 emphasizes the usefulness of molecular traps to promote stereospecific fragment condensation between nonassociating peptide fragments for the synthesis and semisynthesis of polypeptides.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D008722:Methods; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases / Q000032:analysis* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D012260:Ribonucleases; C022542:ribonuclease S; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C019913:clostripain