Institute of Molecular Enzyme Technology, Heinrich -Heine -University Düsseldorf, Forschungszentrum Jülich, D-52426 Jülich, Germany.
Institute of Molecular Enzyme Technology, Heinrich -Heine -University Düsseldorf, Forschungszentrum Jülich, D-52426 Jülich, Germany; Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52428 Jülich, Germany.
J Biotechnol. 2017 Sep 20;258:110-116. doi: 10.1016/j.jbiotec.2017.05.024. Epub 2017 Jun 12.
The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.
大规模的蛋白质工业生产需要有效的分泌，革兰氏阳性菌的 Sec 系统就提供了这种功能。优化分泌的蛋白质工程方法通常涉及到对大型文库的筛选，例如，将目标蛋白与许多不同的信号肽融合。相应的高通量筛选方法通常基于分光光度法或荧光法，能够快速、简单地测定酶活性。在这里，我们报告了一种基于 GFP 分割测定法的替代方法，用于定量分泌蛋白。我们通过测定 GFP 荧光和酶活性分析了枯草芽孢杆菌分泌同源脂肪酶和异源角质酶的情况。此外，我们从一个信号肽文库中鉴定出枯草芽孢杆菌蛋白膨胀素 EXLX1 的变体，其分泌量增加了 5 倍。我们的结果表明，GFP 分割测定法可用于高通量监测枯草芽孢杆菌中酶和非酶蛋白的分泌。
