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 produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modification. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of filamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.
Chang
T K
TK
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Jackson
D Y
DY
Burnier
J P
JP
Wells
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D013381:Subtilisins