Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.
Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.
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产生真核蛋白的表达平台：基于大肠杆菌细胞和小麦生殖细胞的合成，亲和力和溶解度标签以及克隆策略的比较。
DOI：
10.1007/s10969-015-9198-1
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发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Fox BG
中科院分区：
文献类型：
--
作者：
Aceti DJ;Bingman CA;Wrobel RL;Frederick RO;Makino S;Nichols KW;Sahu SC;Bergeman LF;Blommel PG;Cornilescu CC;Gromek KA;Seder KD;Hwang S;Primm JG;Sabat G;Vojtik FC;Volkman BF;Zolnai Z;Phillips GN Jr;Markley JL;Fox BG
设计用于在大肠杆菌中生产蛋白质和小麦无生殖细胞翻译的载体，在结构基因组学管道中选择21种特征明确的真核蛋白作为对照，对其进行了测试。对照通过克隆、小规模表达试验、大规模生长或合成和纯化进行。成功纯化的蛋白质也进行结晶试验或1H-15N HSQC核磁共振分析。实验评价：(1)限制性/结扎和重组克隆系统的相对有效性；(2)麦芽糖结合蛋白（MBP）作为溶解度增强标签的价值；(3) MBP融合的体内蛋白水解作为纯化后蛋白水解的替代方案的后果；(4) LacI抑制因子水平对自诱导大肠杆菌蛋白产量的影响；(5)从无细胞系统产生的蛋白质中去除His标签的后果；(6)大肠杆菌细胞与小麦无生殖细胞翻译的比较性能。讨论了每种表达系统的最佳启动子/阻遏子和融合标签配置。
Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or 1H-15N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.
影响因子：
2.9
作者：
Bae, Euiyoung;Bitto, Eduard;Bingman, Craig A.;McCoy, Jason G.;Wesenberg, Gary E.;Phillips, George N., Jr.
通讯作者：
Phillips, George N., Jr.