Monday, August 11, 2008
P86

An efficient methanol-inducible Pichia pastoris system for expression and secretion of the Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP)

Kin-Ho Law, Yuk-Ki Wong, Ming-San Tsang, Man-Wah Tsang, Pui-Yee Lau, Yun-Chung Leung, and Kwok-Ping Ho. Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China

The β-lactamase inhibitory protein (BLIP), from the Gram-positive bacterium Streptomyces clavuligerus, has been shown to be a potent inhibitor of bacterial class A β-lactamases in many studies. Here, a methanol-inducible BLIP expression system based on the yeast Pichia pastoris was developed for BLIP expression for the first time. In the flask culture, the recombinant BLIP produced by this system was secreted into the culture medium and the purity of this fully active BLIP was >80% even without any purification steps. We have developed an efficient production process in which concentrations of 2-3% of methanol during expression induction produced the best results. The BMGY growth medium was chosen to give higher cell density and hence higher BLIP production. The total time of cultivation was similar to that of the native host S. clavuligerus, but an unprecedented high yield of recoverable protein in culture supernatant (~300 mg of >80% pure BLIP/L culture) was achieved, which is ~20-fold higher than that of ~15 mg/L in S. clavuligerus. In the fermenter culture, ~300 mg/L crude proteins, but only half of which was BLIP, was obtained. Heterologous gene expression was tightly controlled and no production of BLIP was observed before the methanol-induction, suggesting that cell density can be further increased to improve protein yield. An inhibition constant (Ki) value of 0.55 ± 0.07 nM was measured for the interaction between BLIP and the E. coli TEM-1 b-lactamase, similar to the value found for that of the native S. clavuligerus-produced BLIP.

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