兔辅酶Ⅱ依赖性视黄醇脱氢-还原酶功能性表达、纯化及活性鉴定.docVIP

兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶功能性表达、纯化及活性鉴定 　【摘要】 目的：用原核蛋白表达系统对兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NRDR)进行功能性表达，并对其活性进行鉴定。方法：从兔肝中克隆出NRDR全长序列，运用Gateway表达系统将其编码区序列构建到表达载体(pDEST 17)上，再转化到大肠杆菌(E.coli)(BL21_AI)中表达出兔NRDR蛋白并鉴定其表达形式；取工程菌裂解上清通过金属离子亲和层析纯化出目的蛋白，再运用反相高效液相色谱法(HPLC)测定该酶的Km值和Vmax值。结果：构建出含兔NRDR编码区(768bp)的表达克隆，转化到BL21_AI中表达出氨基端含6×His标签的重组兔NRDR蛋白，诱导表达4h后目的蛋白可占总蛋白量的414%；经一步亲和层析得到的兔NRDR纯度为972%，比活性提高了64倍；经HPLC测定，该酶对视黄醛的Km值为(455±033)μmol/L，Vmax为(0307±0065)μmol/(min·mg)。结论：应用pDEST 17可在BL21_AI中对兔肝NRDR进行高效的功能性表达。 【关键词】 辅酶Ⅱ依赖性视黄醇脱氢/还原酶 表达 纯化 类视黄醇代谢 ［Abstract］Objective: To express the rabbit NADP(H)_dependent retinol dehydrogenase/reductase(NRDR)in prokaryotic expression system functionally, and to identify its enzyme activity.Methods: The total NRDR sequence was cloned from the rabbit liver and the coding region of NRDR was constructed to the Gateway_based expression vector (pDEST 17), which was transformed into the Escherichia coli(E.coli)(BL21_AI)for the protein expression. After sonication and centrifugation of the bacteria, the soluble NRDR in supernatant was purified by affinity chromatography. Furthermore, the kinetic parameters of NRDR were determined by high performance liquid chromatography(HPLC).Results: The expression vector with the desired gene(768 bp)was constructed, and then, the NRDR protein, which was harvested at the optimal time point(4 hours after induction), was successfully expressed with a 414% expression level. Moreover, the homogeneous enzyme was obtained by a one_step affinity chromatography, with purity up to 972% and specific activity 64 fold enhanced. The values of the Km and the Vmax were(455±033)μmol/L and(0307±0065)μmol/(min·mg), respectively. Conclusion: The functional rabbit NRDR can be expressed effectively in BL21_AI by pDEST 17. ［Key Words］NADP(H)_dependent retinol dehydrogenase/reductase；expression；purification；retinoids metabolism 维甲酸是脊椎动物体内重要的生理活性物质，通过调节基因的转录参与细胞生长、形态发生、细胞分化、免疫功能调节等生理过程［1,2］。维甲酸在体内是由视黄醇(维生素A)经两步代谢而成，中间代谢产物为视黄醛，即视黄醇视黄醛视黄酸。辅酶Ⅱ依赖性视黄醇脱氢/还原