变形链球菌组氨酸蛋白激酶VicK原核表达、纯化及其蛋白活性检测

Prokaryotic expression,purification and protein activity of histidinekinase VicK of Streptococcus mutans

目的原核表达及纯化变形链球菌组氨酸蛋白激酶VicK,并进行蛋白活性的检测。方法以变形链球菌UA159菌株基因组DNA为模板,PCR扩增VicK基因,插入表达载体pET-28a中,转化大肠埃希菌(E.coli)BL21(DE3)中,IPTG(终浓度为0.01、0.02、0.05、0.1、0.2及0.3 mmol/L)于18及37℃诱导10、15、20 h,表达产物经Ni离子亲和层析柱纯化,采用Kinase-Glo誖激酶发光检测试剂盒检测纯化后目的蛋白的活性。结果重组表达质粒pET-28a-VicK经双酶切及测序鉴定,证明构建正确;表达的重组VicK蛋白相对分子质量约51 000;IPTG终浓度0.3 mmol/L,18℃诱导15 h,可溶性目的蛋白的表达量最高;纯化的目的蛋白纯度>90%,浓度为2 mg/ml;随着VicK蛋白浓度的增加,发光值逐渐降低,表明该蛋白具有体外水解ATP的激酶活性。结论已成功原核表达并纯化了变形链球菌具有激酶活性的VicK蛋白,为后续以此为靶点的抑制物的筛选奠定了实验基础。

Objective To express the histidinekinase VicK of Streptococcus mutans in prokaryotic cells, purify the ex-pressed product and determine its protein activity. Methods The VicK gene was amplified by PCR using the genomic DNA of S. mutans UA159 strain as a template, and inserted into expression vector pET-28a. The constructed recombinant plasmid pET-28a-VicK was transformed to E. coli BL21 （DE3） and induced with IPTG at final concentrations of 0. 01, 0. 02, 0. 05, 0. 1, 0. 2 and 0. 3 mmol/L at 18 and 37 ℃ for 10, 15 and 20 h separately. The expressed product was purified by nickel ion affinity chromatography and determined for protein activity by Kinase-Glo R luminescent kinase as-say. Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a-VieK was constructed cor-rectly. The relative molecular mass of expressed VicK protein was about 51 000. The expression level of soluble target protein reached a peak value after induction with IPTG at a final concentration of 0. 3 mmol/L at 18 ℃ for 15 h. The pu-rity and concentration of purified target protein were more than 90% and 2 mg / ml respectively. The luminescence level of VicK decreased with the increasing concentration, indicating kinase activity of the protein in hydrolysis of ATP in vitro. Conclusion The VieK protein with kinase activity was successfully expressed and purified, which provided an experi-mental basis for further screening of inhibitors targetomg VicK protein of S. mutans.