Mayer Janine, Knuuti Tobias, Baumgarten Lisa, Menke Elise, Bischoff Lena, Bunk Boyke, Biedendieck Rebekka
Institute of Microbiology and Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Rebenring 56, 38106 Braunschweig, Germany.
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7, 38124 Braunschweig, Germany.
Microorganisms. 2022 Apr 5;10(4):777. doi: 10.3390/microorganisms10040777.
The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, provides a promising alternative to common species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in , could be enhanced even up to 29-fold. Overall, only around 170 recombinant clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for and also other species facilitates the identification of suitable SPs for secretion of recombinant proteins.
重组蛋白的分泌在其经济生产和纯化过程中起着重要作用。分泌效率取决于与目标蛋白以及特定宿主相结合的负责信号肽（SP），目前尚无法预测。由于其高质粒稳定性、缺乏碱性胞外蛋白酶且胞外宿主蛋白污染极少，为常见物种提供了一种有前景的替代方案。为了开发一种简便快速的克隆和筛选系统，以鉴定最适合特定蛋白的信号肽，建立了一个基于质粒的信号肽文库，其中包含来自[具体来源]的所有预测的182个依赖Sec的信号肽。将信号肽分成10组单独的多信号肽质粒（pMSP），便于它们进行分组扩增并应用于筛选方法。通过将已分泌良好的α-淀粉酶AmyE的量提高1.6倍，证明了整个文库的功能。一种新型青霉素G酰化酶，由于其天然信号肽不适用于[具体宿主]中的分泌，在细胞内以不溶性蛋白形式存在，其分泌量甚至可提高至29倍。总体而言，仅需筛选约170个基于50个插入信号肽的重组克隆，就能获得足够量用于进一步的酶学特性研究。因此，这种新开发的基于质粒的遗传工具适用于[具体宿主]以及其他[相关宿主]物种，有助于鉴定用于重组蛋白分泌的合适信号肽。
