毛白杨4-香豆酸:辅酶A连接酶可溶性原核表达及活性检测

Soluble prokaryotic expression of 4-coumarate: coenzyme A ligase from Populus tomentosa and enzyme activity of recombinant 4CL1

为了进一步研究毛白杨4CL1的酶学特性，构建了毛白杨4CL1原核表达载体pQE31-4CL1．经IPTG诱导在大肠杆菌中表达了毛白杨4CL1蛋白，SDS-PAGE电泳分析表明该新蛋白的分子量为60kD，与预测值一致．对毛白杨4CL1蛋白在大肠杆菌中的表达体系进行了优化，确定IPTG的最佳浓度为0．4mmol／L，诱导时的菌液密度OD600为0．3-0．5，最佳表达时间为2h．37℃下表达的4CL1融合蛋白以包涵体的形式存在，没有生物学活性．当表达温度从37℃降低为28℃时，可溶性的有生物学活性的毛白杨4CL1蛋白诱导表达获得成功，表达量达11％．以耦联有Ni^2＋-NTA的琼脂糖为亲和层析填料，金属鳌合亲和柱层析一步纯化获得了电泳纯4CL1蛋白，4CL1蛋白对5种底物的比活性分别为：4-香豆酸3949．0pkat／mg，咖啡酸2214．0pkat／mg，阿魏酸715．0pkat／mg，肉桂酸84．9pkat／mg，而对芥子酸没有生物活性．

In order to investigate thoroughly the enzymatic properties of 4-coumarate： coenzyme A ligase （4CL1） from Populus tomentosa, the prokaryotic expression vector of 4CL1 gene pQE31-4CL1 was constructed and transformed into E. coli M15 （pREP4）. The recombinant 4CL1 fused protein was expressed successfully in M15 （pREP4） after M15 （pREP4） was induced by IPTG for 4 h. SDS-PAGE revealed that the mass of the induced protein was about 60 kD consistent with the predicted value. The expression system of the 4CL1 gene in E. coll was optimised, showing that the most effective concentration of IPTG was 0.4 mmol/L, the OD600 of bacteria density of E. coli ranged from about 0.3 to 0.5 and the best expression time was 2 h. The 4CL1 fused proteins existed almost in the form of inclusion bodies expressed at 37℃. The soluble 4CL1 protein which was expressed at 28℃ was active to several substrates. Agarose coupled with Ni^2＋ -NTA was the filling of the metal affinity column chromatography; the electrophoresis purity 4CL1 protein was acquired after one-step purification. The enzymatic activity ratio of the electrophoresis purity 4CL1 to the five substrates, respectively, as follows： 3 949.0 pkat/mg to 4-coumarate, 2 214.0 pkat/mg to caffeic acid, 715.0 pkat/mg to ferulate, 84.9 pkat/mg to cinnamate, no activity to sinapate.