School of Food Science and Engineering, School of Biological and Pharmaceutical Sciences, Shaanxi University of Science and Technology, Xi'an 710021, China.
Xi'an Key Laboratory of Antiviral and Antimicrobial-Resistant Bacteria Therapeutics Research, Xi'an Shaanxi 710021, China.
J Agric Food Chem. 2024 Nov 6;72(44):24632-24642. doi: 10.1021/acs.jafc.4c06174. Epub 2024 Oct 28.
is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.
是一种常用于生产异源蛋白的平台。细胞内表达的重组蛋白的提取和纯化依赖于高效的细胞破碎。为了便于下游处理，设计了可控自溶细胞，当受到内部或外部信号触发时，细胞会自动裂解以释放细胞内蛋白质。在必须采用弱启动子来控制自溶的情况下，即使存在表面活性剂或其他助剂，细胞裂解和产物释放的速度也很慢。在这项研究中，我们报告了一种改进的自溶菌株，该菌株由弱启动子控制，效率更高，无需使用任何促进化学物质。通过柔性肽接头，用带有两亲性穿膜肽的 N 端融合物诱导阿拉伯糖表达 T4 溶菌酶，从而启动细胞裂解。此外，还单独删除了参与膜通透性的基因，并对渗漏表型进行了筛选。（编码 Braun 脂蛋白）的缺失与自溶系统结合，在诱导 4 小时后导致 96%的细胞裂解，在诱导 10 小时后分别释放 84%或 67%的 mCherry 或超大 Cas13a 融合蛋白（160.8 kDa）。这种自溶渗漏菌株在蛋白质回收和文库筛选方面具有很大的应用前景。
