期刊
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
卷 25, 期 7, 页码 1007-1014出版社
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1501.01056
关键词
Antimicrobial peptide; antimicrobial activity; fusion protein; plectasin; prokaryotic expression; penicillin-resistant bacteria
资金
- Combinational Project of Guangdong Province
- Education Ministry for Manufacturing, Education Research [2010B090400540]
- National College Students' Innovation and Entrepreneurship Training Program [201212121080]
- Guangdong Provincial Science and Technology Project [2011B080701062]
- Guangdong Province College Students' Innovative Experiment Project [1212111003]
Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudo plectania 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 genesplicing 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 S-trartsferase 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.
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