Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process

Abstract

SUBTILISIN E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein¹. The pre-sequence functions as the signal peptide for protein secretion across the membrane². Deletion of the pro-sequence yields mature but inactive subtilisin³: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer⁴. We have constructed an active-centre mutant of pro-subtilisin (Asp 32 → Asn)³ which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented⁴. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.