Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.
Department of Biotechnology and Biomedicine, Technical University of Denmark (DTU), Kongens Lyngby, Denmark.
Sci Rep. 2023 Sep 1;13(1):14361. doi: 10.1038/s41598-023-41559-7.
The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli. Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum. To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins.
本研究描述了一种使用 pSIP 系统和植物乳杆菌作为表达宿主表达和纯化整合膜蛋白 RseP 的详细程序。RseP 是一种膜结合的位点 2 蛋白酶，是多种人类病原体中已知的抗菌靶标。在本研究中，我们从革兰氏阳性菌中筛选了五个 RseP 同源物，发现来自屎肠球菌的 RseP（EfmRseP）产生的蛋白水平最高。优化了生产条件，并通过固定化金属离子亲和层析和分子筛层析对 EfmRseP 进行了纯化。纯化后，每 3 克湿重细胞沉淀可获得约 1 毫克纯蛋白。使用圆二色性确认了纯化蛋白的结构完整性。我们进一步评估了屎肠球菌来源的 RseP 在革兰氏阴性大肠杆菌中的表达和纯化。在测试的三种大肠杆菌菌株中，有两种未能检测到可溶性蛋白。与在植物乳杆菌中表达的 EfmRseP 相比，在大肠杆菌 C43(DE3)中表达的 EfmRseP 纯化后的纯度较低。据我们所知，这是首次将植物乳杆菌和 pSIP 表达系统应用于膜蛋白的生产。
