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. 1985 Dec 20;13(24):8913–8926. doi: 10.1093/nar/13.24.8913

Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis.

M Jacobs, M Eliasson, M Uhlén, J I Flock
PMCID: PMC318961  PMID: 3001653

Abstract

The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.

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Selected References

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

  1. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  2. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. GUNTELBERG A. V., OTTESEN M. Purification of the proteolytic enzyme from Bacillus subtilis. C R Trav Lab Carlsberg Chim. 1954;29(3-4):36–48. [PubMed] [Google Scholar]
  4. Gryczan T., Shivakumar A. G., Dubnau D. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol. 1980 Jan;141(1):246–253. doi: 10.1128/jb.141.1.246-253.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kato S., Ishii H., Kano S., Hagihara S., Todoroki T., Nagata S., Takahashi H., Shigeta Y., Tsuchiya M. Alcohol dehydrogenase: a new sensitive marker of hepatic centrilobular damage. Alcohol. 1985 Jan-Feb;2(1):35–38. doi: 10.1016/0741-8329(85)90011-4. [DOI] [PubMed] [Google Scholar]
  6. Keay L., Moser P. W. Differentiation of alkaline proteases from Bacillus species. Biochem Biophys Res Commun. 1969 Mar 10;34(5):600–604. doi: 10.1016/0006-291x(69)90780-3. [DOI] [PubMed] [Google Scholar]
  7. Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984 Jul;12(1):19–36. doi: 10.1016/0147-619x(84)90063-5. [DOI] [PubMed] [Google Scholar]
  8. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Löfdahl S., Guss B., Uhlén M., Philipson L., Lindberg M. Gene for staphylococcal protein A. Proc Natl Acad Sci U S A. 1983 Feb;80(3):697–701. doi: 10.1073/pnas.80.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  12. Nedkov P., Oberthür W., Braunitzer G. Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus. Biol Chem Hoppe Seyler. 1985 Apr;366(4):421–430. doi: 10.1515/bchm3.1985.366.1.421. [DOI] [PubMed] [Google Scholar]
  13. Nilsson B., Holmgren E., Josephson S., Gatenbeck S., Philipson L., Uhlen M. Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci. Nucleic Acids Res. 1985 Feb 25;13(4):1151–1162. doi: 10.1093/nar/13.4.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olsson A., Moks T., Uhlén M., Gaal A. B. Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient. J Biochem Biophys Methods. 1984 Nov;10(1-2):83–90. doi: 10.1016/0165-022x(84)90053-8. [DOI] [PubMed] [Google Scholar]
  15. Rydén A. C., Lindberg M., Philipson L. Isolation and characterization of two protease-producing mutants from Staphylococcus aureus. J Bacteriol. 1973 Oct;116(1):25–32. doi: 10.1128/jb.116.1.25-32.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Smith E. L., DeLange R. J., Evans W. H., Landon M., Markland F. S. Subtilisin Carlsberg. V. The complete sequence; comparison with subtilisin BPN'; evolutionary relationships. J Biol Chem. 1968 May 10;243(9):2184–2191. [PubMed] [Google Scholar]
  18. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  19. Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Takkinen K., Pettersson R. F., Kalkkinen N., Palva I., Söderlund H., Käriäinen L. Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J Biol Chem. 1983 Jan 25;258(2):1007–1013. [PubMed] [Google Scholar]
  21. Uhlén M., Guss B., Nilsson B., Gatenbeck S., Philipson L., Lindberg M. Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J Biol Chem. 1984 Feb 10;259(3):1695–1702. [PubMed] [Google Scholar]
  22. Uhlén M., Guss B., Nilsson B., Götz F., Lindberg M. Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci. J Bacteriol. 1984 Aug;159(2):713–719. doi: 10.1128/jb.159.2.713-719.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vasantha N., Thompson L. D., Rhodes C., Banner C., Nagle J., Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. doi: 10.1128/jb.159.3.811-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wells J. A., Ferrari E., Henner D. J., Estell D. A., Chen E. Y. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 1983 Nov 25;11(22):7911–7925. doi: 10.1093/nar/11.22.7911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wong S. L., Price C. W., Goldfarb D. S., Doi R. H. The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1184–1188. doi: 10.1073/pnas.81.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. doi: 10.1016/0022-2836(84)90192-x. [DOI] [PubMed] [Google Scholar]
  27. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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