The characterization, cloning and overexpression of BLIP gene in Pichia pastoris and Bacillus subtilis

The beta-lactamase inhibitory protein (BLIP), from the Gram-positive bacterium Streptomyces clavuligerus, is a potent inhibitor of bacterial class A beta-lactamases. Overexpressing the GC-rich blip gene in E. coli systems has been a challenge in biotechnology and a limiting factor for obtaining BLIP for studies. Here, we developed an efficient methanol-inducible BLIP expression system based on the yeast Pichia pastoris for the first time. The recombinant BLIP was secreted into culture medium and this fully active BLIP was >80% pure even without any purification steps. Expression induction was optimized with 2-3% methanol. BMGY growth medium gave higher cell density and hence higher BLIP production. The total time of cultivation was similar to that of 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 S. clavuligerus (∼15 mg/L). P. pastoris X-33 was found to be superior for BLIP expression and more than 500 mg/L secreted proteins were found in the supernatant of fed-batch fermentation. However, the production of other cellular proteins of P. pastoris increased with the cell density and at the end of the fermentation constituted more than 50% of the total secreted proteins. Continuous culture strategy was studied. At dilution rate of 0.015-0.02, ∼100 mg/L of BLIP with ∼80% purity was obtained in about 7 days after methanol induction.;Molecular weight of the P. pastoris-expressed BLIP measured by ESI-MS was 18212.72 +/- 3.96, consistent with the calculated value. An inhibition constant (Ki) values of 0.55 +/- 0.07 nM (flask culture); 0.30 +/- 0.06 nM (fed-batch fermenter culture) and 0.61 +/- 0.12 nM (the continuous fermentation culture) were obtained for the interaction between BLIP and the E. coli TEM-1 beta-lactamase, similar to the value (∼0.6 nM) found for that of the native S. clavuligerus-produced BLIP. The growth of a recombinant Bacillus subtilis strain (with both PenP and PenPC beta-lactamase genes) was not inhibited by BLIP alone. However, in the presence of ampicillin, there was no observable growth of the Bacillus in all the cultures with 2.5 muM BLIP protein within 18 hours. This indicates that the BLIP in combinations with the beta-lactam antibiotic - ampicillin was effective in killing the beta-lactamase producing strain. Different protein chromatography methods were used in purifying the recombinant BLIP. However, results showed that purification of BLIP with these methods were not satisfactory. Purification protocol based on the tangential flow filtration (TFF) was finally developed and ∼90% pure BLIP was obtained.