Schimek Clemens, Egger Esther, Tauer Christopher, Striedner Gerald, Brocard Cécile, Cserjan-Puschmann Monika, Hahn Rainer
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Biopharma Process Science, Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria.
Biotechnol Prog. 2020 Sep;36(5):e2999. doi: 10.1002/btpr.2999. Epub 2020 Apr 13.
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.
在这项工作中，我们试图确定一种可在工业规模上应用的选择性提取周质内源性表达蛋白的方法。为此，我们使用了一种表达模型，该模型允许共表达两种荧光蛋白，每种荧光蛋白都特异性地靶向细胞质或周质。我们评估了多种可扩展的裂解方法（高压匀浆、渗透休克程序、用乙二胺四乙酸提取和用脱氧胆酸盐提取），以确定它们选择性提取周质蛋白而非细胞质蛋白的能力。我们的主要结论是，尽管我们确定了可在工业上扩展的裂解条件，这些条件显著提高了进一步纯化的起始纯度，但所测试的条件中没有一个对周质蛋白比对细胞质蛋白具有选择性。此外，我们证明了表达的重组蛋白的有效提取在很大程度上取决于细胞中的总蛋白浓度。
