Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/33365
Title: An efficient heat-inducible Bacillus subtilis bacteriophage φ105 expression and secretion system for the production of the Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP)
Authors: Liu, HB
Chui, KS
Chan, CL
Tsang, CW
Leung, YC 
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)
Issue Date: 2004
Source: Journal of biotechnology, 2004, v. 108, no. 3, p. 207-217 How to cite?
Journal: Journal of Biotechnology 
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 (K i=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.
URI: http://hdl.handle.net/10397/33365
ISSN: 0168-1656
DOI: 10.1016/j.jbiotec.2003.12.004
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