期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 147, 期 -, 页码 76-82出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2018.03.001
关键词
Antimicrobial peptide; Antibacterial activity; Defensin; Pichia pastoris; Stability; Hemolytic activity
类别
资金
- National Natural Science Foundation of China [31501544]
- International Center for Genetic Engineering and Biotechnology [CRP/CHN15-01]
- Natural Science Foundation of Tianjin City [17JCQNJC14400]
- Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education
- Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science Technology) [2016IM002]
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 6.9 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 60 mu g per 1 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 50 mu g/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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