An efficient heat-inducible Bacillus subtilis bacteriophage φ105 expression and secretion system for the production of the Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP)

Hong Bing Liu, Ka Shun Chui, Chi Leong Chan, Chun Wai Tsang, Yun Chung Leung

Research output: Journal article publicationJournal articleAcademic researchpeer-review

9 Citations (Scopus)

Abstract

The Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A β-lactamases including the Escherichia coli TEM-1 β-lactamase (Ki=0.6nM). A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage φ105. The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active. We have shown that the signal peptide of BLIP functions well in B. subtilis to secrete BLIP out of the cells, which facilitates purification. The absence of a His-tag also avoids the activity and structure of BLIP being altered. An unprecedented high yield of recoverable protein in culture supernatant (3.6mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol. We have developed an efficient production process in which the culture time before heat-induction was 3-4h and the culture supernatant could be collected 5h after induction. This total time of 8-9h is considered to be very short compared to that of the native S. clavuligerus culturing (60-70h). We achieved a very efficient BLIP production rate of 0.8-0.9mg/l/h. Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.
Original languageEnglish
Pages (from-to)207-217
Number of pages11
JournalJournal of Biotechnology
Volume108
Issue number3
DOIs
Publication statusPublished - 18 Mar 2004

Keywords

  • Cat
  • Chloramphenicol acetyl transferase gene
  • Chloramphenicol resistance
  • Chloramphenicol sensitive
  • Cm R
  • Cm S
  • Er R
  • Er S
  • ErmC
  • Erythromycin resistance
  • Erythromycin sensitive
  • Kb
  • Kilobase(s)

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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