Sang Ming, Wei Hui, Zhang Jiaxin, Wei Zhiheng, Wu Xiaolong, Chen Yan, Zhuge Qiang
Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
Protein Expr Purif. 2017 Dec;140:44-51. doi: 10.1016/j.pep.2017.08.001. Epub 2017 Aug 4.
ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein.
ABP-dHC-天蚕素A是一种具有抗菌特性的线性阳离子肽。为了探索利用甲基营养型酵母毕赤酵母表达ABP-dHC-天蚕素A的新方法，我们将ABP-dHC-天蚕素A基因克隆到载体pPICZαA中。然后通过电穿孔将经SacI线性化的质粒pPICZαA-ABP-dHC-天蚕素A转化到毕赤酵母GS115中。在含有2%酪蛋白氨基酸的培养基中于20℃用0.5%甲醇培养96小时后诱导表达，以避免蛋白水解。在这些条件下，约48毫克ABP-dHC-天蚕素A分泌到1升（4×250毫升）培养基中。使用尺寸排阻色谱法纯化重组ABP-dHC-天蚕素A，从1升（4×250毫升）培养物中获得21毫克纯的活性ABP-dHC-天蚕素A。在4-20%梯度凝胶上进行电泳表明，重组ABP-dHC-天蚕素A以大小约为4 kDa的蛋白质形式分泌。重组ABP-dHC-天蚕素A成功表达，因为该产物显示出与合成蛋白无法区分的抗菌和抗真菌活性（基于抗菌测定、扫描电子显微镜和抗真菌测定）。
