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Subtiligase: a Tool for Semisynthesis of Proteins THOMAS K Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12544-12548, December 1994 Biochemistry Subtiligase: A tool for semisynthesis of proteins THOMAS K. CHANG*t, DAVID Y. JACKSON*, JOHN P. BURNIERt, AND JAMES A. WELLS*§ Departments of *Protein Engineering and tBioOrganic Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080 Communicated by Harry B. Gray, August 1, 1994 ABSTRACT A variant of subtilisin BPN', which we call phenylalanylamide (glc-F-amide) were synthesized as de- subtiligase, has been used to ligate esterified peptides site- scribed (15). For ligations onto immobilized supports, pep- specifically onto the N termini of proteins or peptides in tides were synthesized on 96-well (---0.17 nmol per well) aqueous solution and in high yield. We have produced biotin- CovaLink ELISA plates (Nunc) by using N-(9-fluorenyl- ylated or heavy-atom derivatives ofmethionyl-extended human methoxycarbonyl) (Fmoc)-protected amino acids and dicyc- growth hormone (Met-hGH) by ligating it onto synthetic pep- lohexylcarbodiimide (DCC) activation in dimethyl sulfoxide tides containing biotin or mercury. Polyethylene glycol (PEG)- (DMSO). The plates were washed with 5% piperidine in modified atrial natriuretic peptide (ANP) was produced by methanol to neutralize the amino linkers and incubated with ligating ANP onto peptides containing sites for PEG modifi'ca- 100 /,u of DMSO containing 1 mM Fmoc-Ala and 0.5 mM tion. We have established the N-terminal sequence require- DCC for 10 min at 25°C. The plates were washed with DMSO, ments for efficient ligation onto proteins, using either synthetic and the Fmoc protecting groups were removed with 5% substrates or pools of ifiamentous phage containing Met-hGH piperidine in methanol. These steps were repeated to gener- with random N-terminal sequences (substrate phage). To fa- ate all 400 natural tripeptides having the form Xaa-Xaa-Ala. cilitate ligations involving proteins with highly structured or Side chains were deprotected by adding 150 ,ul ofCF3COOH/ buried N termini, a more stable subtiligase was designed that anisole, 95:5 (vol/vol) per well for 10 min, and the plates were effectively ligates peptides onto Met-hGH even in 4 M guani- washed three times with 100 mM Tricine (pH 8). dine hydrochloride. The use of subtiligase should expand the To each well was added 100 ,ul of 100 mM Tricine (pH 8) possibilities for protein semisynthesis and rational protein containing 10 ,uM biotinylated Lys-Gly-Ala-Ala-Pro-Phe-glc- design. Phe-amide (bio-KGAAPF-glc-F-amide), followed by addi- tion of 10 ,l of 10 uM subtiligase in Tricine buffer. The ligation proceeded for 15 min at 25°C, and the plate was Coupling of synthetic peptides onto a protein or protein washed with Tricine buffer. SAHRP (Sigma) was diluted fragment is a potentially powerful means of introducing 1:5000 in PBS (10 mM sodium phosphate, pH 7.4/150 mM natural or nonnatural constituents to probe and design pro- NaCl) containing 0.01% Tween and added to the plates (100 tein function (for reviews, see refs. 1-3). For example, ,ul per well), which were incubated for 30 min. Plates were enzymatic and chemical peptide ligation methods have been washed five times with PBS/Tween. o-Phenylenediamine (60 used to produce a variety of semisynthetic proteins (4-14). mg) in 100 ml of 0.1 M citrate/phosphate buffer (pH 5) However, in virtually all cases, coupling relied on noncova- containing 50 ,ul of30% H202 was added (100 ,ul per well). The lent or covalent preassociation or some other special sec- plates were developed for 20 min at 25°C, and the reaction ondary structural feature ofthe fragments to be ligated. Thus, was quenched by the addition of0.5 M H2SO4 (50 ,ul per well). a more general method for the semisynthesis of any protein The absorbance at 405 nm was read with an ELISA plate would be useful. reader. Recently, a variant of the serine protease subtilisin BPN', Substrate Phage Selections. Phagemid phGHam-g3 (17) was in which the catalytic Ser-221 was converted to cysteine and modified to add a methionine codon at the mature N terminus Pro-225 was converted to alanine, was shown to ligate of hGH. The first three residues of Met-hGH were random- dipeptides onto tetrapeptide esters with high kcat values (20 ized with an oligonucleotide containing NNS codons, where s'1) and with little hydrolysis of the ester substrate or N represents all four bases and S represents G or C, by using proteolysis of the amide product (15). Here, we evaluate the site-directed mutagenesis (18). Phagemid particles were pre- sequence requirements for efficient peptide ligation using this pared from Escherichia coli strain XL1-Blue (Stratagene) (19) double mutant of subtilisin BPN', termed "subtiligase," and by using helper phage M13KO7 (20). About 1011 phage were design more stable variants of it. These studies suggest that bound to avidin-conjugated agarose (Pierce) in buffer A subtiligase may be generally useful for site-specific ligation of (phosphate-buffered saline containing 0.5% bovine serum peptides containing affinity handles, isotopic labels, heavy albumin) to filter out the sequences that bind directly to atoms, or other nonnatural constituents onto the N terminus avidin or the matrix. The remaining phage were incubated of proteins or protein fragments. with 0.4 ,uM subtiligase and 1 mM iminobiotin-KGAAPF- glc-F-amide for 1 hr at room temperature. The iminobiotin- MATERIALS AND METHODS conjugated peptide was used because the biotin-avidin in- Materials. Enzymes for DNA manipulations were from teraction is too strong to release under gentle elution condi- New England Biolabs or BRL. Streptavidin-horseradish tions (21). Phage were precipitated twice by the addition of peroxidase (SAHRP) was from GIBCO/BRL, column resins were from Pharmacia, and atrial natriuretic peptide (ANP) Abbreviations: hGH, human growth hormone; A2 or A8 hGH, hGH was from Bachem. Oligonucleotides were synthesized by the with deletions of the first two or eight residues; ANP, atrial natri- Oligonucleotide Synthesis Group at Genentech. uretic peptide; PEG, polyethylene glycol; glc-F-amide, glycol- Peptide Synthesis. All peptides were synthesized by stan- conjugated phenylalanyl amide; NHS, N-hydroxysuccinimide; dard methods (16). Peptides esterified with glycol-conjugated SAHRP, streptavidin-horseradish peroxidase; bio-KGAAPF- and suc-KKKGAAPF-, biotinylated Lys-Gly-Ala-Ala-Pro-Phe- and suc- cinylated Lys-Lys-Lys-Gly-Ala-Ala-Pro-Phe-. The publication costs of this article were defrayed in part by page charge tPresent address: Division of Biotechnology, Syntex Discovery payment. This article must therefore be hereby marked "advertisement" Research, 3401 Hillview Avenue, Palo Alto, CA 94303. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 12544 Downloaded by guest on September 26, 2021 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12545 Table 1. Nucleophile specificity for subtiligase P1i P2' M F K L R A S H W Y G C 1 V T Q N P D E Avg ~~~ ~ .. .. ...... T -19: 1.612 1.4 1.1 0.9 i 1 0.09- 0.8 0.6 0.6 0.3 0.5 02 0.3 03 0.5 0.84 K15.- i- 1. .1 0.7 0.2 0.4 0.4 0.2 0.2 0.2 0.1 0. 0.80 V P~~~~~~~~~.e..412 1.2 1.1. 1. 0.9 0.91-z 0.8 0.6 1 1. 0.5 0.6 0.4 0.5 03 03 0.2 0.5 0.80 H .2.. 1...2.1.0 13.13. 0.8 0.9 0.8 0.4 0.3 0.6 0.3 0.3 0.2 0.3 0.3 0.3 0.73 Y 1.1'}'''''0.9 12'' 1. I 1:2< 0.50.7'O0.8'2 0:7 EII 0.5 0.4 0:2 03 0.3 0.1 0:3 0.2 0:70 A ....1...6- 0.91... 1.0 ...........1.21.00.6 0.7..0.1.2 14-30.7 0.61.0 0.8'1: 0.8Li 0.90.7 0.40.50.20.50.50.4 0.4 0.2 0.20.30.20.207.................0.2 0.2 0.2 0. 0.66 M 0.9 0. 1.0 1...5. 0.7 7 0.5 0.5 0.97 03 0 .3 0.1 0.3 0.2 0.066 D 1-.12:----t -10.92--1.1.1.0 0.6 10.9 0.70 0.8 0.9 0.3 0. 0 0.3 0.3 0.3 0.3 0.3 0.65 F 0.8 1040.5 0.2 0.5 0.9 0.5 0.3 0.2 0.1 0 0.1 0.1 0.63 L 13- 0.9 +1.0-1.01. 1.1 0.7 0.90.7 0.7 0.7 0. 0.4 0.1 0.3 0 0.1 0.1 0.58 W :gt 0.50.9 0.7 0.7 0.5 0.7 0.6 0.6 .LL 0.5 10 053 03 0.2 0.2 0.1 0.2 0.4 0.3 0.56 E 0.8 ELSi 0.9 1l.O 1.0: 0.5 0.7 0.8 0.7 0.6 11.01- 0.3 0.2 0.1 0.6 03 0 0o3 0.2 0.2 0.55 Q 0.8 0.9 0.9 0.9 0.8 0.5 0.7 0.8 0.7 0.6 0.4 0.3 0.4 0.4 0 0.5 0.1 0.2 0.54 G 1.1 0.7 0.8 05 0.8 1.5 0.8 0.8 0.2 0.2 0.6 0.5 0.2 0.2 0.5 0.4 043 0.4 03 0.1 0.54 P 0.8 0.8 0.6 0.6 0.6 0 6 0.9 0.6 0.1 0.2 0.5 0.3 .1 03 0.1 0.2 0 0.1 0.2 0.1 0.38 C 0.2 0*3 0.3 0.2 0.6 0.6 0.2 0.6 0.2 0.2 0.2 0.1 0.3 0.1 0.1 0.1 0.1 0 0.4 0.1 0.24 Avg 1.1 1.0 1.0 1.0 0.9 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 The extent of ligation with bio-KGAAPF-glc-Phe-amide for 400 immobilized tripeptides having the sequence H2N.P1'.
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