链球菌溶血素与真菌果胶酶融合表达及纯化条件的优化  

Optimization of condition for fusion expression and purification of streptolysin O and fungus pectin lyase

作　　者：王建[1,2] 赵庆新[2] 

机构地区：[1]南京工业大学食品与轻工学院,江苏南京211800 [2]盐城师范学院生命科学与技术学院,江苏盐城224051

出　　处：《中国生物制品学杂志》2016年第3期252-256,共5页Chinese Journal of Biologicals

摘　　要：目的优化链球菌溶血素（streptolysin O,SLO）与真菌果胶酶（pectin lyase,PL）的融合表达及纯化条件。方法将活化培养的工程菌以2%接种量接种于发酵液体培养基中,于37℃,200 r/min培养至不同生长期（A_（600）值分别为0.6、0.8和1.0）,分别于不同温度（10、15、20、25、30、35和37℃）及加入不同终浓度IPTG（0.05、0.1、0.2、0.4、0.5、0.6、0.8和1.0 mmol/L）条件下诱导表达不同时间（6、12、20、24、36、48和60 h）。将诱导表达的融合蛋白进行Ni离子亲和层析纯化,对纯化中的上样缓冲液（binding buffer,分别含0、5、10、15、20、25、30 mmol/L咪唑）、清洗缓冲液（washing buffer,分别含0、5、10、20、30、40、50、60 mmol/L咪唑）及洗脱缓冲液（elution buffer,分别含100、200、500、1 000 mmol/L咪唑）的咪唑浓度进行优化。结果最佳诱导表达条件为：工程菌培养至A_（600）为0.8时,加入0.4 mmol/L IPTG,于20℃诱导24 h,目的蛋白的相对分子质量约91 500,占全菌总蛋白的38.6%,溶血活性可达4.0×10~7U/ml。最佳纯化条件为：binding buffer和washing buffer最佳咪唑浓度为5和60 mmol/L,elution buffer分别使用200和500 mmol/L咪唑进行阶段性梯度洗脱,纯化产物纯度可达95%。结论成功优化了SLO与PL融合蛋白的表达及纯化条件,纯化后获得了纯度较高的融合蛋白,为进一步研究SLO蛋白的性质及应用奠定了基础。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.

关 键 词：链球菌溶血素 真菌果胶酶 融合蛋白 诱导表达 纯化 

分 类 号：Q786[生物学—分子生物学]