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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 prokaryotic express and purify Streptococcus mutans histidine protein kinase VicK, and to detect the protein activity. Methods The genomic DNA of Streptococcus mutans UA159 was used as a template to amplify the VicK gene. The gene was inserted into expression vector pET-28a and transformed into E. coli BL21 (DE3). IPTG (final concentration 0.01, 0.02 0.05, 0.1, 0.2 and 0.3 mmol / L) were induced at 18 and 37 ℃ for 10, 15 and 20 h respectively. The expressed product was purified by Ni ion affinity chromatography and purified with Kinase-Glo paradox kinase kit The activity of the target protein. Results The recombinant plasmid pET-28a-VicK was identified by double enzyme digestion and sequencing. The recombinant protein was confirmed to be correctly constructed. The relative molecular mass of the expressed recombinant VicK protein was about 51 000. The final concentration of IPTG was 0.3 mmol / L, and induced by 18 ℃ for 15 h. The purity of the purified protein was> 90% at a concentration of 2 mg / ml. With the increase of the concentration of VicK protein, the luminescence decreased gradually, indicating that the protein has the kinase activity of hydrolyzing ATP in vitro. Conclusions Prokaryotic expression and purification of VicK protein of kinase-active Streptococcus mutans have been successfully established, which laid the foundation for the follow-up screening of inhibitors.