High-Level Expression Of The Antimicrobial Peptide Plectasin In Escherichia Coli

Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella found on the floor of northern European pine forests. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially the Staphylococcus aureus and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In vitro, Plectasin could kill Staphylococcus aureus and Streptococcus pneumoniae rapidly at rates comparable to both vancomycin and penicillin and did not exhibit cross-resistance with other conventionally used antibiotics. Plectasin showed no cytotoxic effects on mammalian cell viability. With its excellent CSF penetration reaching33%, being higher than other conventional antibiotics, and potent bactericidal activity in CSF, plectasin could be a promising new option for the treatment of central nervous system infection.This study to describe the expression of plectasin in E. coli expression system and its antibacterial activity. In this study, the coding sequence of plectasin was obtained from NCBI GenBank and optimized according to the codon usage table of E. coli. We designed four primers to amplify the fragment by PCR-based gene SOEing synthesis, and then cloned into pET32a (+) vector. The fusion protein was expressed in Escherichia coli with IPTG induction, and then purified by Ni2+-chelating affinity chromatography. The mature plectasin was excised from fusion protein by Factor Xa protease cleavage, and its antibacterial activity was also investigated by a radial diffusion assay.In this study the results showed that the Recombinant plasmid pET32a-PLEC was constructed and was expressed solubly at a high yield reaching about89%of the total bacteria protein with0.8mmol/L IPTG induction for3h. The recombinant fusion protein was purify by Ni2+-chelating affinity chromatography reaching an apparent purity of97%, and then cleaved by Factor Xa. The radial diffsion assay results validated that plectasin has a high bactericidal activity against Staphylococcus aureus comparable to ampicillin. These works might provide a significant foundation for the following production or study of plectasin, and contribute to the development and evolution of novel antimicrobial drugs in clinical applications.