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. 1997 Feb 1;321(Pt 3):699–705. doi: 10.1042/bj3210699

The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli.

Y C Leung 1, C Chan 1, J S Reader 1, A C Willis 1, R J van Spanning 1, S J Ferguson 1, S E Radford 1
PMCID: PMC1218125  PMID: 9032456

Abstract

The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to approximately 80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.

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Selected References

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  1. Adman E. T. Copper protein structures. Adv Protein Chem. 1991;42:145–197. doi: 10.1016/s0065-3233(08)60536-7. [DOI] [PubMed] [Google Scholar]
  2. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  3. Arai H., Igarashi Y., Kodama T. The structural genes for nitric oxide reductase from Pseudomonas aeruginosa. Biochim Biophys Acta. 1995 Apr 4;1261(2):279–284. doi: 10.1016/0167-4781(95)00018-c. [DOI] [PubMed] [Google Scholar]
  4. Arvidsson R. H., Nordling M., Lundberg L. G. The azurin gene from Pseudomonas aeruginosa. Cloning and characterization. Eur J Biochem. 1989 Jan 15;179(1):195–200. doi: 10.1111/j.1432-1033.1989.tb14540.x. [DOI] [PubMed] [Google Scholar]
  5. Berks B. C., Baratta D., Richardson J., Ferguson S. J. Purification and characterization of a nitrous oxide reductase from Thiosphaera pantotropha. Implications for the mechanism of aerobic nitrous oxide reduction. Eur J Biochem. 1993 Mar 1;212(2):467–476. doi: 10.1111/j.1432-1033.1993.tb17683.x. [DOI] [PubMed] [Google Scholar]
  6. Berks B. C., Richardson D. J., Reilly A., Willis A. C., Ferguson S. J. The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. Biochem J. 1995 Aug 1;309(Pt 3):983–992. doi: 10.1042/bj3090983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan C., Willis A. C., Robinson C. V., Aplin R. T., Radford S. E., Ferguson S. J. The complete amino acid sequence confirms the presence of pseudoazurin in Thiosphaera pantotropha. Biochem J. 1995 Jun 1;308(Pt 2):585–590. doi: 10.1042/bj3080585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen J. Y., Chang W. C., Chang T., Chang W. C., Liu M. Y., Payne W. J., LeGall J. Cloning, characterization, and expression of the nitric oxide-generating nitrite reductase and of the blue copper protein genes of Achromobacter cycloclastes. Biochem Biophys Res Commun. 1996 Feb 15;219(2):423–428. doi: 10.1006/bbrc.1996.0249. [DOI] [PubMed] [Google Scholar]
  9. Chou M. M., Kendall D. A. Polymeric sequences reveal a functional interrelationship between hydrophobicity and length of signal peptides. J Biol Chem. 1990 Feb 15;265(5):2873–2880. [PubMed] [Google Scholar]
  10. Doud S. K., Chou M. M., Kendall D. A. Titration of protein transport activity by incremental changes in signal peptide hydrophobicity. Biochemistry. 1993 Feb 9;32(5):1251–1256. doi: 10.1021/bi00056a008. [DOI] [PubMed] [Google Scholar]
  11. Errington J. A rapid and reliable one-step method for isolating DNA fragments from agarose gels. Nucleic Acids Res. 1990 Sep 11;18(17):5324–5324. doi: 10.1093/nar/18.17.5324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frew J. E., Hill H. A. Direct and indirect electron transfer between electrodes and redox proteins. Eur J Biochem. 1988 Mar 1;172(2):261–269. doi: 10.1111/j.1432-1033.1988.tb13882.x. [DOI] [PubMed] [Google Scholar]
  13. Hoitink C. W., Woudt L. P., Turenhout J. C., van de Kamp M., Canters G. W. Isolation and sequencing of the Alcaligenes denitrificans azurin-encoding gene: comparison with the genes encoding blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis. Gene. 1990 May 31;90(1):15–20. doi: 10.1016/0378-1119(90)90434-s. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  15. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  16. Moir J. W., Baratta D., Richardson D. J., Ferguson S. J. The purification of a cd1-type nitrite reductase from, and the absence of a copper-type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor. Eur J Biochem. 1993 Mar 1;212(2):377–385. doi: 10.1111/j.1432-1033.1993.tb17672.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Silvestrini M. C., Galeotti C. L., Gervais M., Schininà E., Barra D., Bossa F., Brunori M. Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Lett. 1989 Aug 28;254(1-2):33–38. doi: 10.1016/0014-5793(89)81004-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Spiro S., Guest J. R. Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol. 1987 Dec;133(12):3279–3288. doi: 10.1099/00221287-133-12-3279. [DOI] [PubMed] [Google Scholar]
  21. Stoll R., Page M. D., Sambongi Y., Ferguson S. J. Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis. Microbiology. 1996 Sep;142(Pt 9):2577–2585. doi: 10.1099/00221287-142-9-2577. [DOI] [PubMed] [Google Scholar]
  22. Van Spanning R. J., De Boer A. P., Reijnders W. N., Spiro S., Westerhoff H. V., Stouthamer A. H., Van der Oost J. Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators. FEBS Lett. 1995 Feb 27;360(2):151–154. doi: 10.1016/0014-5793(95)00091-m. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Williams P. A., Fülöp V., Leung Y. C., Chan C., Moir J. W., Howlett G., Ferguson S. J., Radford S. E., Hajdu J. Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase. Nat Struct Biol. 1995 Nov;2(11):975–982. doi: 10.1038/nsb1195-975. [DOI] [PubMed] [Google Scholar]
  25. Yamamoto K., Uozumi T., Beppu T. The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells. J Bacteriol. 1987 Dec;169(12):5648–5652. doi: 10.1128/jb.169.12.5648-5652.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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