A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides sitespecifically 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 modifi'cation. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of ifiamentous 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.Coupling of synthetic peptides onto a protein or protein fragment is a potentially powerful means of introducing natural or nonnatural constituents to probe and design protein function (for reviews, see refs. 1-3). For example, enzymatic and chemical peptide ligation methods have been used to produce a variety of semisynthetic proteins (4-14). However, in virtually all cases, coupling relied on noncovalent or covalent preassociation or some other special secondary structural feature ofthe fragments to be ligated. Thus, a more general method for the semisynthesis of any protein would be useful.Recently, a variant of the serine protease subtilisin BPN', in which the catalytic Ser-221 was converted to cysteine and Pro-225 was converted to alanine, was shown to ligate dipeptides onto tetrapeptide esters with high kcat values (20 s'1) and with little hydrolysis of the ester substrate or proteolysis of the amide product (15). Here, we evaluate the sequence requirements for efficient peptide ligation using this double mutant of subtilisin BPN', termed "subtiligase," and design more stable variants of it. These studies suggest that subtiligase may be generally useful for site-specific ligation of peptides containing affinity handles, isotopic labels, heavy atoms, or other nonnatural constituents onto the N terminus of proteins or protein fragments.MATERIALS AND METHODS Materials. Enzymes for DNA manipulations were from New England Biolabs or BRL. Streptavidin-horseradish peroxidase (SAHRP) was from GIBCO/BRL, column resins were from Pharmacia, and atrial natriuretic peptide (ANP) was from Bachem. Oligonucleotides were synthesized by the Oligonucleotide Synthesis Group at Genentech.Peptide Synthesis. All peptides were synthesized by standard methods (16). Peptides esterified with glycol-conjugated phenylalanylamide (glc-F-amide) were synthesized as described (15). For ligations onto immobilized supports, peptides were synthesized on 96-well (---0.17 nmol p...