Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.

中文翻译：

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用于在大肠杆菌中进行蛋白质亚细胞定位研究的可靠分级分离方法。

革兰氏阴性细菌的分级分离用于鉴定蛋白质的亚细胞定位，特别是输出的重组蛋白质的定位。细胞分离的过程可能充满交叉污染问题，并且通常缺乏有关分离纯度的支持数据。在这里，我们比较了三种周质提取和两种细胞破碎技术的不同组合，以研究哪个过程产生了大肠杆菌的未污染区室。从这些数据中，可以编译出一种名为PureFrac的可靠方法，该方法可以得到纯的周质级分，并且可以回收可溶性细胞质蛋白。该方法在用超声和超速离心将细胞质与不溶性物质分离之前，先用冷渗透镁溶液提取周质。