Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark

• Published in: Scientific reports 2023-09, Vol.13 (1), p.14361-14361, Article 14361
• Main Authors: Kristensen, Sofie S., Lukassen, Marie V., Siebenhaar, Suzana, Diep, Dzung B., Morth, J. Preben, Mathiesen, Geir
• Format: Article
• Language: English
• Subjects: 631/1647; 631/1647/2230; 631/1647/2230/1378; 631/1647/2230/2233; 631/326; 631/337; 631/45; 631/535; 631/61; 631/92; Affinity chromatography; Bacteria; Chromatography; Circular dichroism; E coli; Enterococcus faecium; Escherichia coli; Gram-positive bacteria; Humanities and Social Sciences; Integral membrane proteins; Lactiplantibacillus plantarum; Membranes; Metal ions; multidisciplinary; Protein purification; Proteins; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• 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.