人重组烟酰胺单核苷酸腺苷转移酶2的表达、纯化及酶活力测定

Expression,purification and determination of enzyme activity of human recombinant nicotinamide mononucleotide Adenylyltransferase-2

目的:通过大肠杆菌体系诱导表达烟酰胺单核苷酸腺苷酰转移酶2(NMNAT2),并测定纯化的重组NMNAT2酶蛋白催化烟酰胺腺嘌呤二核苷酸(NAD)生物合成的酶比活力。方法:在大肠杆菌BL21(DE3)体系优化条件下诱导表达NMNAT2重组蛋白,利用Ni-NTA Superflow Kit进行目的蛋白的纯化,Western blot法进行鉴定,并通过酶联荧光法测定其催化产物NAD,测定其酶活性。结果:人重组质粒NMNAT2-p ET-24a可以在大肠杆菌BL21(DE3)体系中诱导表达。诱导条件为:LB培养液(卡那霉素,15μg/m L)中37℃,IPTG(1.0 m M)诱导表达4 h。纯化获得的NMNAT2重组蛋白具有较高的酶活性。结论:NMNAT2-p ET-24a可在大肠杆菌BL21(DE3)体系中稳定表达,优化条件下可获得较高活性的纯化NMNAT2重组蛋白,可用于神经保护功能及酶蛋白的活性调控研究。

Objective To induce the expression of nicotinamide mononucleotide adenylyltransferase-2 （ NMNAT2） through E.coli system ,and to determine the enzyme specific activity of purified recombinant NMNAT2 apoenzyme in catalyzing the biosynthesis ofnicotinate adenine dinucleotide （ N A D ）. Methods：Under the optimized condition of E. coli BL21 （ DE3 ） system, NMNAT2 recombinantprotein was induced to express. The target protein was purified using Ni-NTA Superflow K it,and was indentified using Western blotmethod. B esides,its catalysate NAD was measured by enzyme-linked assay,and the enzyme actively was also determined. Results：Thehuman recombinant plasmid construct NMNAT2-pET-24a could be induced to express in E. coli BL21 （D E 3） system. The inductionconditions were ： LB broth （ kanam ycin, 15 （jig/mL） in 37 °C , IPTG （ 1. 0 mM ） for 4 hours. And the purified recombinant proteinNMNAT2 had high enzyme activity. Conclusion： NMNAT2-pET-24a can be expressed stably in E. coli BL21 （D E 3） system. The purifiedrecombinant protein NMNAT2 with higher activity can be obtained under the optimized condition, which can be applied in the studyof neuroprotective function and regulation of apoenzyme activity.