Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Dec 20;91(26):12544–12548. doi: 10.1073/pnas.91.26.12544

Subtiligase: a tool for semisynthesis of proteins.

T K Chang 1, D Y Jackson 1, J P Burnier 1, J A Wells 1
PMCID: PMC45475  PMID: 7809074

Abstract

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.

Full text

PDF
12544

Images in this article

12546Fig. 1
on p.12546

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abrahmsén L., Tom J., Burnier J., Butcher K. A., Kossiakoff A., Wells J. A. Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution. Biochemistry. 1991 Apr 30;30(17):4151–4159. doi: 10.1021/bi00231a007. [DOI] [PubMed] [Google Scholar]
  2. Bongers J., Lambros T., Liu W., Ahmad M., Campbell R. M., Felix A. M., Heimer E. P. Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor. J Med Chem. 1992 Oct 16;35(21):3934–3941. doi: 10.1021/jm00099a022. [DOI] [PubMed] [Google Scholar]
  3. Chaiken I. M. Semisynthetic peptides and proteins. CRC Crit Rev Biochem. 1981;11(3):255–301. doi: 10.3109/10409238109108703. [DOI] [PubMed] [Google Scholar]
  4. Chang M. S., Lowe D. G., Lewis M., Hellmiss R., Chen E., Goeddel D. V. Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature. 1989 Sep 7;341(6237):68–72. doi: 10.1038/341068a0. [DOI] [PubMed] [Google Scholar]
  5. Chen K., Arnold F. H. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5618–5622. doi: 10.1073/pnas.90.12.5618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chinkers M., Garbers D. L. Signal transduction by guanylyl cyclases. Annu Rev Biochem. 1991;60:553–575. doi: 10.1146/annurev.bi.60.070191.003005. [DOI] [PubMed] [Google Scholar]
  7. Cunningham B. C., Wells J. A. Comparison of a structural and a functional epitope. J Mol Biol. 1993 Dec 5;234(3):554–563. doi: 10.1006/jmbi.1993.1611. [DOI] [PubMed] [Google Scholar]
  8. Cunningham B. C., Wells J. A. Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. Protein Eng. 1987 Aug-Sep;1(4):319–325. doi: 10.1093/protein/1.4.319. [DOI] [PubMed] [Google Scholar]
  9. Devlin J. J., Panganiban L. C., Devlin P. E. Random peptide libraries: a source of specific protein binding molecules. Science. 1990 Jul 27;249(4967):404–406. doi: 10.1126/science.2143033. [DOI] [PubMed] [Google Scholar]
  10. Fuh G., Cunningham B. C., Fukunaga R., Nagata S., Goeddel D. V., Wells J. A. Rational design of potent antagonists to the human growth hormone receptor. Science. 1992 Jun 19;256(5064):1677–1680. doi: 10.1126/science.256.5064.1677. [DOI] [PubMed] [Google Scholar]
  11. Gaertner H. F., Offord R. E., Cotton R., Timms D., Camble R., Rose K. Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor. J Biol Chem. 1994 Mar 11;269(10):7224–7230. [PubMed] [Google Scholar]
  12. Gráf L., Li C. H. Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6135–6138. doi: 10.1073/pnas.78.10.6135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heney G., Orr G. A. The purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B. Anal Biochem. 1981 Jun;114(1):92–96. doi: 10.1016/0003-2697(81)90456-5. [DOI] [PubMed] [Google Scholar]
  14. Homandberg G. A., Laskowski M., Jr Enzymatic resynthesis of the hydrolyzed peptide bond(s) in ribonuclease S. Biochemistry. 1979 Feb 20;18(4):586–592. doi: 10.1021/bi00571a006. [DOI] [PubMed] [Google Scholar]
  15. Jackson D. Y., Burnier J., Quan C., Stanley M., Tom J., Wells J. A. A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science. 1994 Oct 14;266(5183):243–247. doi: 10.1126/science.7939659. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  17. Lowman H. B., Bass S. H., Simpson N., Wells J. A. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry. 1991 Nov 12;30(45):10832–10838. doi: 10.1021/bi00109a004. [DOI] [PubMed] [Google Scholar]
  18. Matthews D. J., Wells J. A. Substrate phage: selection of protease substrates by monovalent phage display. Science. 1993 May 21;260(5111):1113–1117. doi: 10.1126/science.8493554. [DOI] [PubMed] [Google Scholar]
  19. McPhalen C. A., James M. N. Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo. Biochemistry. 1988 Aug 23;27(17):6582–6598. [PubMed] [Google Scholar]
  20. Morihara K. Enzymatic semisynthesis of human insulin: an update. J Mol Recognit. 1990 Oct-Dec;3(5-6):181–186. doi: 10.1002/jmr.300030502. [DOI] [PubMed] [Google Scholar]
  21. Narhi L. O., Stabinsky Y., Levitt M., Miller L., Sachdev R., Finley S., Park S., Kolvenbach C., Arakawa T., Zukowski M. Enhanced stability of subtilisin by three point mutations. Biotechnol Appl Biochem. 1991 Feb;13(1):12–24. [PubMed] [Google Scholar]
  22. Pantoliano M. W., Whitlow M., Wood J. F., Dodd S. W., Hardman K. D., Rollence M. L., Bryan P. N. Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. Biochemistry. 1989 Sep 5;28(18):7205–7213. doi: 10.1021/bi00444a012. [DOI] [PubMed] [Google Scholar]
  23. Roy R. P., Khandke K. M., Manjula B. N., Acharya A. S. Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction. Biochemistry. 1992 Aug 18;31(32):7249–7255. doi: 10.1021/bi00147a007. [DOI] [PubMed] [Google Scholar]
  24. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  25. Sealock R. W., Laskowski M., Jr Thermodynamics and kinetics of the reactive site peptide-bond hydrolysis in bovine pancreatic secretory trypsin inhibitor (Kazal). Biochemistry. 1973 Aug 14;12(17):3139–3146. doi: 10.1021/bi00741a001. [DOI] [PubMed] [Google Scholar]
  26. Simmerman H. K., Wang C. C., Horwitz E. M., Berzofsky J. A., Gurd F. R. Semisynthesis of sperm whale myoglobin by fragment condensation. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7739–7743. doi: 10.1073/pnas.79.24.7739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  28. Wallace C. J. Understanding cytochrome c function: engineering protein structure by semisynthesis. FASEB J. 1993 Apr 1;7(6):505–515. doi: 10.1096/fasebj.7.6.8386119. [DOI] [PubMed] [Google Scholar]
  29. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES