Cooperative Research Centre for Tissue Growth and Repair, Department of Chemical Engineering, The University of Adelaide, SA 5005, Australia.
Biotechnol Bioeng. 1997 Mar 5;53(5):453-8. doi: 10.1002/(SICI)1097-0290(19970305)53:5<453::AID-BIT2>3.0.CO;2-G.
Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet-raacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453-458, 1997.
从大肠杆菌中提取细胞内蛋白质传统上是通过机械破坏来实现的。本研究考察了一种破坏细菌细胞壁完整性的化学处理方法，该方法可能提供一种替代技术。用螯合剂乙二胺四乙酸（EDTA）（大于 0.3 mM）和变性剂脲（6 M）的组合处理在从未诱导的大肠杆菌中释放蛋白质方面非常有效。在 3 mM EDTA 存在下的 6 M 脲可以从对数期和静止期大肠杆菌细胞中以相当于机械破坏的水平释放细胞质蛋白。两种化学试剂的浓度是影响最大蛋白质释放水平的主要变量。还确定了几个较小的变量和相互作用。蛋白质释放动力学是一级的。对于 2、4 和 6 M 脲和 3 mM EDTA，时间常数约为 2.5 分钟，与脲浓度无关。没有脲的 3 mM EDTA 的动力学要慢得多，时间常数为 12.3 分钟。（c）1997 年 John Wiley & Sons，Inc.生物工程 53:453-458，1997。
