Letter to the Editor:: Solution structure of a homodimeric hypothetical protein, At5g22580, a structural genomics target from Arabidopsis thaliana
Letter to the Editor:: Solution structure of a homodimeric hypothetical protein, At5g22580, a structural genomics target from Arabidopsis thaliana
复制标题
DOI：
10.1023/b:jnmr.0000032525.70677.16
复制
发表时间：
2004-07-01
影响因子：
2.7
通讯作者：
Markley, JL
中科院分区：
文献类型：
--
作者：
Cornilescu, G;Cornilescu, CC;Markley, JL
方法和结果将At5g22580基因克隆到pET15b （Novagen, Madison， WI）中，位于NdeI和BamHI酶切位点之间。该构建体编码含有凝血酶可切割（thrombincleable, His） 6标签的19个氨基酸的n端融合，并在T7启动子的控制下表达。将质粒转化为大肠杆菌Rosetta (DE3)/pLysS菌株（Novagen, Madison， WI）。细胞在含有[15N]-氯化铵（用于15N-蛋白）或[15N]-氯化铵和[U-13C]-葡萄糖（用于生产15N； 13c -蛋白）的最小培养基上生长(Q。赵等，j。功能。染色体组。,接受)。利用BugBuster HT （Novagen）对收获的细胞进行破坏，在Qiagen （Valen-）的Ni-NTA柱上固定化金属亲和层析（IMAC）纯化蛋白质
Methods and resultsThe At5g22580 gene was cloned into pET15b (Novagen, Madison, WI), between the NdeI and BamHI restriction sites. The construct coded for a 19-amino acid N-terminal fusion containing a thrombincleavable (His) 6 tag and provided for expression under the control of a T7 promoter. The plasmid was transformed into E. coli Rosetta (DE3)/pLysS strain (Novagen, Madison, WI). The cells were grown on a minimal medium containing [15N]-ammonium chloride (for 15N-protein) or [15N]-ammonium chloride and [U-13C]-glucose (for the production of 15N; 13C-protein)(Q. Zhao et al., J. Struct. Funct. Genom., accepted). The harvested cells were disrupted using BugBuster HT (Novagen), and the protein was purified by immobilized metal affinity chromatography (IMAC) on a Ni-NTA column from Qiagen (Valen-