AbstractInducible expression system

Abstract
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, 15N or 13C, 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).

Keywords
Hybrid photon detectors; Auto-induction; T7 expression system; Lactose; pBAD promoter; Arabinose; Protein production; High-density batch cultures; Metabolic control of pH; Selenomethionine labeling; Isotopic labeling

結果 (中文) 1:

摘要其中 T7 RNA 聚合酶转录编码序列的 T7lac 启动子的克隆下控制有效的诱导表达系统生产种类繁多的蛋白质在大肠杆菌中。影响稳定、 增长和 T7 表达菌株在摇晃船只导致零星的、 意想不到诱导表达的复杂介质，先前报道的其他人，几乎可以肯定造成的识别由少量的乳糖诱导的因素的调查。葡萄糖会阻止感应的乳糖精心研究的机制。氨基酸也抑制诱导乳糖日志阶段生长过程和曝气的高利率抑制在低乳糖浓度感应。这些意见，和代谢平衡 ph 值，允许发展可靠的非诱导和自动诱导的媒体批文化成长中到高的密度。表达株增加到非诱导介质中饱和度保留质粒，并保持完全可行周在冰箱里，使其易于编写并行的许多冰箱股票和使用工作股票长一段时间。自动感应允许高效筛选的多克隆表达和溶解性，为多机并联作为文化有只接种并将其增至饱和，并通常成倍地高于常规 IPTG 诱导获得的目标蛋白质产率。自动诱导媒体已标记蛋白与硒代蛋氨酸、 15N 或 13 C 和为靶标蛋白生产糖诱导的 T7 RNA 聚合酶在 BL21 AI pBAD 启动子。硒代蛋氨酸贴标是同样有效的常用的蛋氨酸营养缺陷型 （发现要检定） 的 B834(DE3) 或 prototroph 将此等。关键字混合光子探测器 ；自动感应 ；T7 表达系统 ；乳糖 ；pBAD 发起人 ；阿拉伯糖 ；蛋白质生产 ；高密度批文化 ；代谢控制 ph 值 ；硒代蛋氨酸标记 ；同位素标记法

結果 (中文) 3:

摘要
诱导表达系统T7 RNA聚合酶转录，编码克隆的T7lac启动子控制下有效地产生各种各样的蛋白质在大肠杆菌中的序列。对影响稳定性的因素增长，调查，和振动容器LED来识别，零星的T7表达菌株诱导，复杂介质中诱导表达的意外，先前所报道的人，几乎肯定是由少量的乳糖引起的。葡萄糖乳糖的研究机制，防止感应。氨基酸也抑制诱导乳糖在对数生长期，而高水平的曝气抑制在低乳糖浓度诱导。这些观察，和pH代谢平衡，允许开发可靠的非诱导和自动诱导介质中分批培养生长到高密度。表达菌株生长在非诱导培养基中保留质粒和完全可行的周在冰箱的饱和度，使它容易准备许多冷冻股平行使用股票的一段。自动感应允许在并行的表达和溶解性多克隆有效的筛选，为文化必须接种和生长至饱和，与目标蛋白的产率通常高出几倍，比传统的IPTG诱导获得的。自动诱导媒体已开发的标记蛋白质与硒，15N或13C，对目标蛋白的T7 RNA聚合酶的阿拉伯糖诱导PBAD启动子在BL21-AI生产。硒代蛋氨酸甲硫氨酸营养缺陷型标记在常用b834同样有效（DE3）（有量）和BL21（DE3）型。

关键词
混合光子探测器；自动感应；T7表达系统；乳糖；PBAD启动子；糖；蛋白质的生产；高密度培养；pH控制代谢；硒代蛋氨酸标记；同位素标记