Secretion and Periplasmic Activation of a Potent Endonuclease in E. coli

Abstract

AbstractSm Endonuclease (SmEn) is a promiscuous, highly active nuclease widely used in protein purification, 2D protein gels, and gene and cell therapy. We aimed to recombinantly and economically produce this reagent using E. coli. Despite widespread application of E. coli for recombinant production of proteins, cytoplasmic expression of this protein resulted in no activity accumulation. We therefore investigated translocation of SmEn to the periplasm of E. coli by evaluating several signal sequences, E. coli host cells, and incubation conditions. For rapid feedback, we developed a crude lysate-based nuclease activity assay that enabled convenient screening and identified suitable conditions for active SmEn accumulation. Signal sequence selection was most influential with additional benefit gained by slowing synthesis either using the transcriptionally weakened strain, C43 (DE3) or by reducing incubation temperature. While our study provides valuable insights for optimizing a nuclease translocation and reducing production costs, more research is needed to explore the influence of mRNA secondary structure at the translation initiation region on protein expression and translocation. Overall, our rapid screening assay facilitated the development of an effective production process for a protein with potential cytoplasmic toxicity as well as the need of disulfide bond formation.