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. 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.
Jing
Xiao-Lan
XL
Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin, People's Republic of China.
Luo
Xue-Gang
XG
Tian
Wen-Jing
WJ
Lv
Li-Hui
LH
Jiang
Yong
Y
Wang
Nan
N
Zhang
Tong-Cun
TC
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
D023181:Antimicrobial Cationic Peptides; D003062:Codon; D010455:Peptides; D011993:Recombinant Fusion Proteins; C504857:plectasin; D013879:Thioredoxins