High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus
文献类型：期刊论文
| 作者 | Ling, C; Zheng, YG; Wang, ED |
| 刊名 | PROTEIN EXPRESSION AND PURIFICATION
 |
| 出版日期 | 2004 |
| 卷号 | 36期号:1页码:146-149 |
| 关键词 | leucyl-tRNA synthetase argU overproduction Aquifex aeolicus Escherichia coli |
| 通讯作者 | Wang, ED (reprint author), Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, State Key Lab Mol Biol, 320 Yue Yang Rd, Shanghai 200031, Peoples R China.,edwang@sibs.ac.cn |
| 英文摘要 | Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The arg U gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His(6) tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His(6)-tag at the N-terminus did not affect the aminoacylation activity of the enzyme. (C) 2004 Elsevier Inc. All rights reserved. |
| 学科主题 | Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology |
| 类目[WOS] | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 关键词[WOS] | GENES |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000222037200019 |
| 版本 | 出版稿 |
| 源URL | [http://202.127.25.143/handle/331003/2011]  |
| 专题 | 上海生化细胞研究所_上海生科院生化细胞研究所 |
| 推荐引用方式 GB/T 7714 | Ling, C,Zheng, YG,Wang, ED. High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus[J]. PROTEIN EXPRESSION AND PURIFICATION,2004,36(1):146-149. |
| APA | Ling, C,Zheng, YG,&Wang, ED.(2004).High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus.PROTEIN EXPRESSION AND PURIFICATION,36(1),146-149. |
| MLA | Ling, C,et al."High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus".PROTEIN EXPRESSION AND PURIFICATION 36.1(2004):146-149. |
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