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
. 1993 Jan 15;90(2):615–619. doi: 10.1073/pnas.90.2.615

Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli.

J H Laity 1, S Shimotakahara 1, H A Scheraga 1
PMCID: PMC45714  PMID: 8421696

Abstract

Wild-type ribonuclease A and five mutants thereof have been expressed in Escherichia coli as fusion proteins by using a T7 expression system. The five mutants are C[65-72]S, C[40-95]S, C[58-110]S, C[26-84]S, and K41G. The expressed fusion protein formed inclusion bodies which were then cleaved by factor Xa. The cleaved ribonuclease A was isolated as unfolded (sulfonated), soluble protein which was subsequently folded. This expression system can be used to produce mutants of ribonuclease A in yields suitable for folding and structural studies. All four native three-disulfide mutants exhibited enzymatic activity (5-30%), although only two were thermally stable at room temperature, demonstrating that no single native disulfide bond is essential for folding. The K41G mutant was enzymatically inactive with cyclic cytidine monophosphate as substrate.

Full text

PDF
615

Images in this article

616Image
on p.616
617Image
on p.617

Selected References

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

  1. Adler M., Scheraga H. A. Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow. Biochemistry. 1988 Apr 5;27(7):2471–2480. doi: 10.1021/bi00407a033. [DOI] [PubMed] [Google Scholar]
  2. Ahmed A. K., Schaffer S. W., Wetlaufer D. B. Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers. J Biol Chem. 1975 Nov 10;250(21):8477–8482. [PubMed] [Google Scholar]
  3. Allemann R. K., Presnell S. R., Benner S. A. A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity? Protein Eng. 1991 Oct;4(7):831–835. doi: 10.1093/protein/4.7.831. [DOI] [PubMed] [Google Scholar]
  4. Altman J. D., Henner D., Nilsson B., Anderson S., Kuntz I. D. Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin. Protein Eng. 1991 Jun;4(5):593–600. doi: 10.1093/protein/4.5.593. [DOI] [PubMed] [Google Scholar]
  5. CROOK E. M., MATHIAS A. P., RABIN B. R. Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2':3'-phosphate. Biochem J. 1960 Feb;74:234–238. doi: 10.1042/bj0740234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Creighton T. E. Intermediates in the refolding of reduced ribonuclease A. J Mol Biol. 1979 Apr 15;129(3):411–431. doi: 10.1016/0022-2836(79)90504-7. [DOI] [PubMed] [Google Scholar]
  7. Creighton T. E. Kinetics of refolding of reduced ribonuclease. J Mol Biol. 1977 Jun 25;113(2):329–341. doi: 10.1016/0022-2836(77)90145-0. [DOI] [PubMed] [Google Scholar]
  8. Creighton T. E. Toward a better understanding of protein folding pathways. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5082–5086. doi: 10.1073/pnas.85.14.5082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denton J. B., Konishi Y., Scheraga H. A. Folding of ribonuclease A from a partially disordered conformation. Kinetic study under folding conditions. Biochemistry. 1982 Oct 12;21(21):5155–5163. doi: 10.1021/bi00264a008. [DOI] [PubMed] [Google Scholar]
  10. Hantgan R. R., Hammes G. G., Scheraga H. A. Pathways of folding of reduced bovine pancreatic ribonuclease. Biochemistry. 1974 Aug 13;13(17):3421–3431. doi: 10.1021/bi00714a001. [DOI] [PubMed] [Google Scholar]
  11. Konishi Y., Ooi T., Scheraga H. A. Regeneration of RNase A from the reduced protein: models of regeneration pathways. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5734–5738. doi: 10.1073/pnas.79.18.5734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konishi Y., Ooi T., Scheraga H. A. Regeneration of ribonuclease A from the reduced protein. Energetic analysis. Biochemistry. 1982 Sep 14;21(19):4741–4748. doi: 10.1021/bi00262a034. [DOI] [PubMed] [Google Scholar]
  13. Konishi Y., Ooi T., Scheraga H. A. Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment. Biochemistry. 1981 Jul 7;20(14):3945–3955. doi: 10.1021/bi00517a001. [DOI] [PubMed] [Google Scholar]
  14. Konishi Y., Ooi T., Scheraga H. A. Regeneration of ribonuclease A from the reduced protein. Rate-limiting steps. Biochemistry. 1982 Sep 14;21(19):4734–4740. doi: 10.1021/bi00262a033. [DOI] [PubMed] [Google Scholar]
  15. Konishi Y., Scheraga H. A. Regeneration of ribonuclease A from the reduced protein. 1. Conformational analysis of the intermediates by measurements of enzymatic activity, optical density, and optical rotation. Biochemistry. 1980 Apr 1;19(7):1308–1316. doi: 10.1021/bi00548a008. [DOI] [PubMed] [Google Scholar]
  16. Lin S. H., Konishi Y., Denton M. E., Scheraga H. A. Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Conformational and thermodynamic analysis of cross-linked (lysine7-lysine41)-ribonuclease a. Biochemistry. 1984 Nov 6;23(23):5504–5512. doi: 10.1021/bi00318a019. [DOI] [PubMed] [Google Scholar]
  17. Nagai K., Thøgersen H. C. Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. 1984 Jun 28-Jul 4Nature. 309(5971):810–812. doi: 10.1038/309810a0. [DOI] [PubMed] [Google Scholar]
  18. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  19. SELA M., ANFINSEN C. B. Some spectrophotometric and polarimetric experiments with ribonuclease. Biochim Biophys Acta. 1957 May;24(2):229–235. doi: 10.1016/0006-3002(57)90186-5. [DOI] [PubMed] [Google Scholar]
  20. Scheraga H. A., Konishi Y., Ooi T. Multiple pathways for regenerating ribonuclease A. Adv Biophys. 1984;18:21–41. doi: 10.1016/0065-227x(84)90005-4. [DOI] [PubMed] [Google Scholar]
  21. Scheraga H. A., Konishi Y., Rothwarf D. M., Mui P. W. Toward an understanding of the folding of ribonuclease A. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5740–5744. doi: 10.1073/pnas.84.16.5740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schultz D. A., Baldwin R. L. Cis proline mutants of ribonuclease A. I. Thermal stability. Protein Sci. 1992 Jul;1(7):910–916. doi: 10.1002/pro.5560010709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  24. Thannhauser T. W., Scheraga H. A. Reversible blocking of half-cystine residues of proteins and an irreversible specific deamidation of asparagine-67 of S-sulforibonuclease under mild conditions. Biochemistry. 1985 Dec 17;24(26):7681–7688. doi: 10.1021/bi00347a027. [DOI] [PubMed] [Google Scholar]
  25. Vasantha N., Filpula D. Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis. Gene. 1989 Mar 15;76(1):53–60. doi: 10.1016/0378-1119(89)90007-3. [DOI] [PubMed] [Google Scholar]
  26. Vasantha N., Thompson L. D. Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences. J Bacteriol. 1986 Mar;165(3):837–842. doi: 10.1128/jb.165.3.837-842.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wearne S. J., Creighton T. E. Further experimental studies of the disulfide folding transition of ribonuclease A. Proteins. 1988;4(4):251–261. doi: 10.1002/prot.340040404. [DOI] [PubMed] [Google Scholar]
  28. Wlodawer A., Svensson L. A., Sjölin L., Gilliland G. L. Structure of phosphate-free ribonuclease A refined at 1.26 A. Biochemistry. 1988 Apr 19;27(8):2705–2717. doi: 10.1021/bi00408a010. [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