| High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli |
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| 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 |
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| 作者姓名： | Fan Bing-you Lu Hai Jiang Xiang-ning |
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| 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 |
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| 基金项目： | 国家重点基础研究发展计划(973计划) |
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| 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.
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| 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 |
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| 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 |
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| Authors: | Fan Bing-you Lu Hai Jiang Xiang-ning |
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| 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 |
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| 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. |
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| Keywords: | 4-coumarate:coenzyme A ligase Populus tomentosa prokaryotic expression enzyme activity |
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