title: High-level expression of the antimicrobial peptide plectasin in Escherichia coli.
abstract: Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin could kill the sensitive strain as efficaciously as vancomycin and penicillin and without cytotoxic effects on mammalian cell viability. In order to establish a bacterium-based plectasin production system, in the present study, the coding sequence of plectasin was optimized, and then cloned into pET32a (+) vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lysate was separated by Ni(2+)-chelating affinity chromatography. The purified protein was then cleaved by Factor Xa protease to release mature plectasin. Final purification was achieved by Ni(2+)-chelating chromatography again. The recombinant plectasin exhibited the same antimicrobial activity as reported previously. This is the first study to describe the expression of plectasin in E. coli expression system, and 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.
authors.affiliation: Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin, People's Republic of China.
authors.forename: Xiao-Lan
authors.initials: XL
authors.lastname: Jing
authors.forename: Xue-Gang
authors.initials: XG
authors.lastname: Luo
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Tian
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Lv
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Tong-Cun
authors.initials: TC
authors.lastname: Zhang
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D023181:Antimicrobial Cationic Peptides; D003062:Codon; D010455:Peptides; D011993:Recombinant Fusion Proteins; C504857:plectasin; D013879:Thioredoxins