Expression of cecropin B in Pichia pastoris and its bioactivity in vitro

Wang,Xiuqing; Zhu,Mingxing; Yang,Guimao; Su,Chunxia; Zhang,Aijun; Cao,Ruibing; Chen,Puyan

doi:10.3892/etm.2011.262

Expression of cecropin B in Pichia pastoris and its bioactivity in vitro

Authors:
- Xiuqing Wang
- Mingxing Zhu
- Guimao Yang
- Chunxia Su
- Aijun Zhang
- Ruibing Cao
- Puyan Chen
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Affiliations: School of Clinical Laboratory of Medicine, NingXia Medical University, Yinchuan 750004, P.R. China, Laboratory Animal Centre, NingXia Medical University, Yinchuan 750004, P.R. China, School of Basic Medical Science, NingXia Medical University, Yinchuan 750004, P.R. China, Key Laboratory of Animal Disease Diagnosis and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, P.R. China
Published online on: May 9, 2011 https://doi.org/10.3892/etm.2011.262
Pages: 655-660

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Abstract

Natural cecropin B (CB), consisting of 35 amino acids, is a member of the cecropin family with the highest antibacterial activity. Here, a novel approach for the generation of recombinant CB in the methylotrophic yeast Pichia pastoris was explored. For this purpose, the CB gene was amplified by recursive PCR (rPCR) and cloned into the pPICZαA vector. The SacI-linearized plasmid pPICZαA-CB was transformed into P. pastoris SMD1168 by electroporation. The expression of recombinant CB was induced with 1.0% methanol at pH 5.0 for 60 h at 28˚C. Recombinant CB was purified using cationic exchange chromatography; 5.0 mg of pure active CB was obtained from 100 ml of culture broth supernatant. Antimicrobial assays demonstrated that CB has a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional CB for both research and industrial purposes.

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