Huang Miaorong, Chen Ruiai, Ren Guangcai
Key Laboratory of Biotechnology and Drug Manufacture for Animal Epidemic Prevention, Ministry of Agriculture, Zhaoqing, China.
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
PLoS One. 2017 Aug 23;12(8):e0183764. doi: 10.1371/journal.pone.0183764. eCollection 2017.
The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ cell to generate recombinant bacmid carrying Ker-His-Flag. Recombinant viruses were produced by infecting insect Spodoptera frugiperda (Sf9) cells with bacmid DNA and used for proteins production. Target proteins were purified from the cell supernatants by Ni2+-NTA affinity chromatography and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The purified product contained two peptides with molecular weights of 38 kDa and 30 kDa and had an optimal pH and temperature at 8.0 and 45°C for keratinolytic activity, respectively. The final product had a specific activity of about 635 U/mg. In summary, we have demonstrated that the open reading frame containing recombinant Ker-His-Flag was expressed and secreted by leader peptide of mellittin from Apis mellitera in insect cells and affinity purification through 8His-Flag tag. It presents an alternative technology for producing keratinases. To our knowledge, it was the first report on the expression of functional keratinase from Bacillus licheniformis in insect cells system.
地衣芽孢杆菌PWD-1的角蛋白酶（kerA）基因在昆虫细胞中进行了表达和纯化。首先，根据该蛋白质和肽的氨基酸序列设计了编码Ker-His-Flag的序列，并进行了密码子优化，以确保在昆虫细胞中的高表达。下一步，将合成的DNA插入pUC57载体，然后通过BamHI/HindIII酶切位点亚克隆到pFastBac™-1供体载体中。构建的载体转化到大肠杆菌DH10Bac™细胞中，以产生携带Ker-His-Flag的重组杆粒。通过用杆粒DNA感染昆虫草地贪夜蛾（Sf9）细胞来生产重组病毒，并用于蛋白质生产。通过Ni2+-NTA亲和层析从细胞上清液中纯化目标蛋白，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹进行评估。纯化产物包含两种分子量分别为38 kDa和30 kDa的肽，其角蛋白分解活性的最佳pH和温度分别为8.0和45°C。最终产物的比活性约为635 U/mg。总之，我们已经证明，包含重组Ker-His-Flag的开放阅读框在昆虫细胞中由意大利蜜蜂蜂毒肽的前导肽表达和分泌，并通过8His-Flag标签进行亲和纯化。它提供了一种生产角蛋白酶的替代技术。据我们所知，这是关于在地衣芽孢杆菌中功能性角蛋白酶在昆虫细胞系统中表达的首次报道。
