兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶的功能性表达、纯化及活性鉴定

Functional Expression,Purification and Activity Identification of Rabbit NADP(H)-Dependent Retinol Dehydrogenase/Reductase

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(768 bp)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4 h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。

Objective： To express the rabbit NADP（H）-dependent retinol dehydrogenase/reductase（NRDR）in prokaryotie 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 eentrifugation of the bacteria, the soluble NRDR in supematant was purified by affinity chromatography. Furthermore, the kinetic parameters of NRDR were determined by high performance liquid chromatography（HPLC）. Resuits： 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 Krn 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.