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 pe ptide 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 cleav age in the presence of small nucleophilic compounds, releasing the N-termin al extein. With a proper choice of the nucleophilic compounds it is shown, that it is possible to generate long polypeptides, by molecular biology exp ression, with such an attached reactive ester which is an excellent substra te of the enzyme, subtiligase. This approach can successfully extend the cu rrent limit of the subtiligase-catalyzed fragment condensation method as we ll as provide another application of the recently discovered intein chemist ry. (C) 1999 Academic Press.