Use of benzyl mercaptan for direct preparation of long polypeptide benzylthio esters as substrates of subtiligase

Subtiligase, a double mutant of subtilisin, has been shown to be capable of joining together two unprotected peptide fragments, namely an activated peptide ester and a second peptide with a free N-terminal amino group. Inside cells, inteins are know to join peptide chains (exteins) by self-extrusion. The SC VMA1 intein was modified to undergo only in vitro N-terminal cleavage in the presence of small nucleophilic compounds, releasing the N-terminal extein. With a proper choice of the nucleophilic compounds it is shown, that it is possible to generate long polypeptides, by molecular biology expression, with such an attached reactive ester which is an excellent substrate of the enzyme, subtiligase. This approach can successfully extend the current limit of the subtiligase-catalyzed fragment condensation method as well as provide another application of the recently discovered intein chemistry. (C) 1999 Academic Press.