Protein production by auto-induction in high-density shaking cultures
Protein production by auto-induction in high-density shaking cultures
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DOI：
10.1016/j.pep.2005.01.016
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发表时间：
2005-05-01
影响因子：
1.6
通讯作者：
Studier, FW
中科院分区：
文献类型：
--
作者：
Studier, FW
诱导表达系统中，T7 RNA聚合酶转录的编码序列克隆下的T7 lac启动子的控制下，有效地产生各种各样的蛋白质在大肠杆菌。影响T7表达菌株在振荡容器中的稳定性、生长和诱导的因素的调查导致认识到，先前由其他人报道的在复杂培养基中的零星的、非预期的表达诱导几乎肯定是由少量乳糖引起的。葡萄糖通过充分研究的机制防止乳糖诱导。氨基酸也抑制诱导乳糖在对数生长期，高通气率抑制诱导低乳糖浓度。这些观察结果和pH的代谢平衡允许开发可靠的非诱导和自诱导培养基，其中分批培养物生长至高密度。在非诱导培养基中生长至饱和的表达菌株保留质粒，并在冰箱中保持完全存活数周，从而易于平行制备许多冷冻储备液并在延长的时间内使用工作储备液。自动诱导允许平行地有效筛选许多克隆的表达和溶解性，因为培养物仅需接种并生长至饱和，并且靶蛋白的产率通常比通过常规IPTG诱导获得的高几倍。已经开发了自动诱导培养基，用于用硒代蛋氨酸、N-15或C-13标记蛋白质，以及用于通过阿拉伯糖诱导T7 RNA聚合酶从BL 21-AI中的pBAD启动子产生靶蛋白。硒代甲硫氨酸标记在常用的甲硫氨酸营养缺陷型B834（DE 3）（发现是metE）或原养型BL 21（DE 3）中同样有效。爱思唯尔公司出版
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, N-15 or C-13, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3). Published by Elsevier Inc.