title: The Stability, and Efficacy Against Penicillin-Resistant Enterococcus faecium, of the Plectasin Peptide Efficiently Produced by Escherichia coli.
abstract: Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using gene-splicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentration-dependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.
authors: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China.
authors: Xin
authors: X
authors: Chen
authors: Nanfang Hospital, First Clinical Medical College, Southern Medical University, Guangzhou 510515, P.R. China.
authors: Yaoan
authors: Y
authors: Wen
authors: Biosafety Level-3 Laboratory, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China.
authors: Ling
authors: L
authors: Li
authors: Nanfang Hospital, First Clinical Medical College, Southern Medical University, Guangzhou 510515, P.R. China.
authors: Jiawei
authors: J
authors: Shi
authors: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China.
authors: Zhe
authors: Z
authors: Zhu
authors: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China.
authors: Yuwen
authors: Y
authors: Luo
authors: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China.
authors: Yun
authors: Y
authors: Li
authors: Department of Respiratory Diseases, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, P.R. China.
authors: Rui
authors: R
authors: Chen
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004355:Drug Stability; D016984:Enterococcus faecium / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; C504857:plectasin
keywords: Antimicrobial peptide; antimicrobial activity; fusion protein