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High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli
引用本文:Fan Bing-you Lu Hai Jiang Xiang-ning. High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli[J]. 中国林学(英文版), 2007, 9(3): 208-212. DOI: 10.1007/s11632-007-0033-z
作者姓名:Fan Bing-you Lu Hai Jiang Xiang-ning
作者单位:Fan Bing-you1,2 Lu Hai1 Jiang Xiang-ning1* 1College of Biological Sciences and Biotechnology,Beijing Forestry University,Beijing 100083,P. R. China 2College of Agriculture,Henan University of Science and Technology,Luoyang 471003,P. R. China
基金项目:国家重点基础研究发展计划(973计划)
摘    要:In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL1 was transformed into expression host M15 (pREP4) and induced by isopropyl-α-D-thiogalactoside (IPTG) to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol·L-1 IPTG induction as the ex-pression temperature declined from 37 to 28°C. The 6×His tag facilitates affinity binding to Ni2 -nitrolotriacetic acid (NTA) and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose cou-pled with Ni2 -NTA affinity chromatography. The optimal substrate for Pt4CL1 was 4-coumarate.

关 键 词:辅酶 连接酶因子 重组体 蛋白质 木质素 酚聚合体 有脉植物 矢量 脱氧核糖核酸
收稿时间:2007-03-01
修稿时间:2007-03-01

High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli
Fan Bing-you,Lu Hai,Jiang Xiang-ning. High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli[J]. Forestry Studies in China, 2007, 9(3): 208-212. DOI: 10.1007/s11632-007-0033-z
Authors:Fan Bing-you  Lu Hai  Jiang Xiang-ning
Affiliation:1. College of Agriculture, Henan University of Science and Technology, Luoyang 471003, P. R. China;College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, P. R. China
2. College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, P. R. China
Abstract:In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL1 was transformed into expression host M15 (pREP4) and induced by isopropyl-α-D-thiogalactoside (IPTG) to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol·L−1 IPTG induction as the expression temperature declined from 37 to 28°C. The 6×His tag facilitates affinity binding to Ni2+-nitrolotriacetic acid (NTA) and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni2+-NTA affinity chromatography. The optimal substrate for Pt4CL1 was 4-coumarate.
Keywords:4-coumarate:coenzyme A ligase  Populus tomentosa  prokaryotic expression  enzyme activity
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