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

Yun Chung Leung, Christopher Chan, John S. Reader, Anthony C. Willis, Rob J.M. Van Spanning, Stuart J. Ferguson, Sheena E. Radford

Research output: Journal article publicationJournal articleAcademic researchpeer-review

25 Citations (Scopus)

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 ~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.
Original languageEnglish
Pages (from-to)699-705
Number of pages7
JournalBiochemical Journal
Volume321
Issue number3
DOIs
Publication statusPublished - 1 Feb 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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