Crystal structure of Escherichia coli YfhJ protein, a member of the ISC machinery involved in assembly of iron-sulfur clusters
Crystal structure of Escherichia coli YfhJ protein, a member of the ISC machinery involved in assembly of iron-sulfur clusters
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DOI：
10.1002/prot.20481
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发表时间：
2005-08-15
影响因子：
2.9
通讯作者：
Fukuyama, K
中科院分区：
文献类型：
--
作者：
Shimomura, Y;Takahashi, Y;Fukuyama, K
材料和方法：过量生产和纯化：用引物5 ′-GGATCC GGACTTAAGTGGACCGATAG-3 ′和5 ′-GTCGAC TATTATTCGGCCTCGTCCAGC-3 ′通过聚合酶链反应（PCR）扩增YfhJ的编码区，其中下划线序列分别是BamHI和SalI的限制性位点。将PCR产物克隆到pCR 2中。1-使用TA克隆法从TOPO载体（Invitrogen）中扩增，并验证序列。用BamHI和SalI消化质粒，并将片段克隆到pQE-30载体（Qiagen）的相应位点中，以构建表达N-末端（His）6-标记的YfhJ蛋白的pQ-ORF 3质粒。将该质粒导入E. coliM15细胞，其含有pREP 4，携带lacI基因。将细胞在补充有50 μ g/mL氨苄青霉素、20 μ g/mL卡那霉素和0.5mMIPTG的Terrific肉汤中于32 ℃培养10小时.将pQ-ORF3质粒导入E. coliB834（DE3）细胞，以产生硒代蛋氨酸（SeMet）标记的YfhJ蛋白。将细胞在含有SeMet代替甲硫氨酸的基本培养基中于32 ℃培养30小时.通过Ni-NTA（Qiagen）色谱法，随后在Sephacryl S-200高分辨率柱（阿默舍姆Biosciences）上凝胶过滤来纯化（His）6-标记的YfhJ蛋白。
Materials and Methods.Overproduction and purification:The coding region of YfhJ was amplified by polymerase chain reaction (PCR) with the primers 5′-GGATCC GGACTTAAGTGGACCGATAG-3′ and 5′-GTCGAC TATTATTCGGCCTCGTCCAGC-3′, where the underlined sequences are restriction sites of BamHI and SalI, respectively. The PCR product was cloned into the pCR2. 1-TOPO vector (Invitrogen) using a TA cloning method, and the sequence was verified. The plasmid was digested with BamHI and SalI, and the fragment was cloned into the corresponding sites of the pQE-30 vector (Qiagen) to construct the pQ-ORF3 plasmid expressing the N-terminal (His) 6-tagged YfhJ protein. This plasmid was introduced into E. coli M15 cells harboring pREP4, carrying the lacI gene. The cells were cultured at 32 C for 10 h in Terrific broth supplemented with 50 μg/mL ampicillin, 20 μg/mL kanamycin, and 0.5 mM IPTG. The pQ-ORF3 plasmid was also introduced into E. coli B834 (DE3) cells auxotrophic for methionine, to produce the selenomethionine (SeMet)-labeled YfhJ protein. The cells were cultured at 32 C for 30 h in minimal medium containing SeMet instead of methionine. The (His) 6-tagged YfhJ protein was purified by Ni-NTA (Qiagen) chromatography followed by gel filtration on a Sephacryl S-200 High Resolution column (Amersham Biosciences).