In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high‐pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins. Our main conclusion was that although we identified industrially scalable lysis conditions that significantly increased the starting purity for further purification, none of the tested conditions were selective for periplasmic protein over cytoplasmic protein. Furthermore, we demonstrated that efficient extraction of the expressed recombinant proteins was largely dependent on the overall protein concentration in the cell.

中文翻译：

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在工业相关工艺条件下提取重组周质蛋白：选择性和产量在很大程度上取决于蛋白质滴度和方法

在这项工作中，我们试图确定一种选择性提取周质内源表达蛋白的方法，该方法适用于工业规模。为此，我们使用了一种表达模型，该模型允许两种荧光蛋白共表达，每种荧光蛋白都专门针对细胞质或周质。我们评估了许多可扩展的裂解方法（高压匀浆、渗透休克程序、乙二胺四乙酸提取和脱氧胆酸盐提取）选择性提取周质蛋白而不是细胞质蛋白的能力。我们的主要结论是，虽然我们确定了工业规模化裂解条件，显着提高了进一步纯化的起始纯度，没有一种测试条件对周质蛋白的选择性高于细胞质蛋白。此外，我们证明了表达重组蛋白的有效提取在很大程度上取决于细胞中的整体蛋白质浓度。