腺苷同型半胱氨酸水解酶的表达纯化及活性测定方法建立

Expression and purification of adenosyl homocysteine hydrolase and establishment of its activity determination method

目的获得高纯度的重组S-腺苷同型半胱氨酸水解酶(SAHH),并建立其活性测定方法。方法运用基因工程技术,在大肠杆菌系统中原核表达重组SAHH,经过镍亲和层析和Sephadex G15两步纯化,得到目的蛋白对其进行理化性质鉴定,并通过优化活性测定条件,确定SAHH活性测定方法。结果终产物SAHH纯度为95%,相对分子质量为49000,比活性为1615U/mg。在37℃、pH7.5的HEPES缓冲液中反应20min为酶活性测定最佳条件,抑制剂可有效抑制酶活性。结论通过基因重组技术可获得高纯度的SAHH,产物理化性质良好,活性测定方法成功建立,为循环酶法测定同型半胱氨酸工艺稳定放大提供可循依据,并且为SAHH抑制剂科学研究提供一种可靠的方法。

Objective To obtain high purified recombinant S-adenosyl homocysteine hydrolase（SAHH） and establish its activity determination method.Methods Genetic engineering technology was adopted to express recombinant SAHH in E.coli prokaryotic expression system.The target protein was obtained via two-step purification including nickel affinity chromatography and Sephadex G15,and its physical and chemical properties were identified.SAHH activity determination method was established through optimizing the condition of activity measurement.Results The purity of endproducts SAHH was 95% with relative molecular mass of 49 000 and the specific activity of 1 615 U/mg.The condition of 37 ℃,pH7.5 HEPES buffer and 20 minutes＇ reaction was optimum for SAHH activity determination.SAHH inhibitor effectively inhibited its activity.Conclusion High purified SAHH can be obtained through recombinant genetic technology with good physical and chemical propoties.Successful establishment of its activity determination provide a basis for stable amplification of circulating homocysteine enzymatic measurement technology and a reliable method for scientific research on SAHH inhibitors.