Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8359488 | Protein Expression and Purification | 2018 | 25 Pages |
Abstract
VpDef is a novel defensin isolated from the clam Venerupis philippinarum. Previously it was expressed in Escherichia coli; however, the E. coli-derived recombinant VpDef did not show effective antimicrobial activity against Staphyloccocus aureus or the Gram-negative bacteria tested. As such, the goal of this study was to design, express, and purify a recombinant VpDef (rVpDef) in Pichia pastoris and to determine its antibacterial potency and stability. A 6.9â¯KDa rVpDef was successfully expressed as a secreted peptide in P. pastoris, and the amount of rVpDef accumulation was shown to reach as high as approximate 60â¯Î¼g per 1â¯ml of culture medium only after an initial optimization was performed. The purified rVpDef demonstrated a broad antibacterial spectrum and was active against six typical common bacteria, both Gram-positive and Gram-negative. A minimal inhibition concentration of as low as 50â¯Î¼g/ml was observed for rVpDef against the growth of E. coli O157 (ATCC 35150). Moreover, rVpDef was tolerant to temperature shock and proteinase digestion and maintained a high stability over a relatively broad pH range. In addition, rVpDef had a low hemolytic activity against rabbit erythrocytes. Taken together, this study demonstrated that rVpDef could be produced in a large-scale manner in P. pastoris and has a good antibacterial activity and suitable stability. This is the first report on heterologous expression of a biologically active VpDef in P. pastoris, supporting its use for both research and application purposes.
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Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
De-Mei Meng, Yu-Jie Lv, Jing-Fang Zhao, Qing-Yan Liu, Lin-Yue Shi, Jun-Ping Wang, Yong-Hai Yang, Zhen-Chuan Fan,