WANG Jian;ZHAO Qing-xin;College of Food Science and Light Industry, Nanjing University of Technology;Objective To optimize the condition for fusion expression and purification of streptolysin O(SLO)and fungus pectin lyase(PL). Methods The activated E. coli BL21(DE3)cells was inoculated to liquid fermentation medium at a MOI of 2%, and incubated at 37 ℃, 200 r / min until the A_(600) values reached 0. 6, 0. 8 and 1. 0, then induced with IPTG at various final concentrations(0. 05, 0. 1, 0. 2, 0. 4, 0. 5, 0. 6, 0. 8 and 1. 0 mmol / L) and various temperatures(10,15, 20, 25, 30, 35 and 37 ℃)for 6, 12, 20, 24, 36, 48 and 60 h separately. The expressed fusion protein was purified by nickel ion affinity chromatography, based on which the binding buffer(containing 0, 5, 10, 15, 20, 25 and 30 mmol / L imidazole respectively), washing buffer(containing 0, 5, 10, 20, 30, 40, 50 and 60 mmol / L imidazole respectively) and elution buffer(containing 100, 200, 500 and 1 000 mmol / L imidazole respectively) were optimized. Results The optimal condition for expression was as follows: the recombinant E. coli was incubated until th A_(600) value was 0. 8, then induced with 0. 4 mmol / L IPTG at 20 ℃ for 24 h. The expressed target protein, with a relative molecular mass of about91 500, contained 38. 6% of total somatic protein, of which the hemolytic activity was 4. 0 × 10~7 U / ml. The optimal imidazole concentrations in binding and washing buffers were 5 and 60 mmol / L respectively. However, fractional gradient elution with 200 and 500 mmol / L imidazole was adopted. The purified protein reached a purity of 95%. Conclusion The condition for expression and purification of SLO and PL fusion protein was optimized, and highly purified fusion protein was obtained, which laid a foundation of further study on property and application of SLO.
2016 03 v.29 [Abstract][OnlineView][Download 269K]