SUBTILIGASE - A TOOL FOR SEMISYNTHESIS OF PROTEINS

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides site-specifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have p roduced biotinylated or heavy-atom derivatives of methionyl-extended h uman growth hormone (Met-hGH) by ligating it onto synthetic peptides c ontaining biotin or mercury. Polyethylene glycol (PEG)modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides c ontaining sites for PEG modification. We have established the N-termin al sequence requirements for efficient ligation onto proteins, using e ither synthetic substrates or pools of filamentous phage containing Me t-hGH with random N-terminal sequences (substrate phage). To facilitat e ligations involving proteins with highly structured or buried N term ini, a more stable subtiligase was designed that effectively ligates p eptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of s ubtiligase should expand the possibilities for protein semisynthesis a nd rational protein design.