Li Yangyuan, Wu Shuge, Ouyang Jing, Mao Limin, Li Wensheng, Lin Haoran
State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.
Protein Expr Purif. 2012 Jul;84(1):80-5. doi: 10.1016/j.pep.2012.04.019. Epub 2012 May 4.
IGF-1 plays a key role in development, growth, and metabolism in teleost. Recombinant fish IGF-1 may be a useful tool for both theoretical research and aquaculture applications. However, using the Escherichia coli expression system has several drawbacks for producing quality fish IGF-1 protein. To explore the yeast expression system for generating fish IGF-1 protein, the cDNA coding for the mature orange-spotted grouper IGF-1 peptide without signal peptide and E domain was cloned into the secreting expression organism Pichia pastoris. Tricine-SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rgIGF-1 was secreted into the culture medium, had a molecular weight of 8.7 kDa. The production peaked at 24h of induction and the optimal pH for expression was 5.0. The recombinant protein was purified using a combined ammonium sulfate precipitation with Ni(2+) affinity chromatography. Finally, 17.9 mg of the protein was obtained from 420 ml of the culture supernatant and the purity was about 92.4%. Bioactivity of the rgIGF-1 was confirmed by the ability to stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell. The present results suggest that the Pichia pastoris expression system can be used to produce a functional rgIGF-1 for both research and aquaculture application.
胰岛素样生长因子-1（IGF-1）在硬骨鱼的发育、生长和代谢中起关键作用。重组鱼IGF-1可能是理论研究和水产养殖应用的有用工具。然而，使用大肠杆菌表达系统生产高质量的鱼IGF-1蛋白存在几个缺点。为了探索用于生成鱼IGF-1蛋白的酵母表达系统，将编码无信号肽和E结构域的成熟点带石斑鱼IGF-1肽的cDNA克隆到分泌表达生物体巴斯德毕赤酵母中。对甲醇诱导表达酵母克隆的培养基进行的Tricine-SDS-PAGE和蛋白质印迹分析表明，重组IGF-1（rgIGF-1）分泌到培养基中，分子量为8.7 kDa。产量在诱导24小时时达到峰值，表达的最适pH为5.0。重组蛋白通过硫酸铵沉淀与Ni(2+)亲和层析相结合的方法进行纯化。最后，从420 ml培养上清液中获得了17.9 mg的蛋白，纯度约为92.4%。rgIGF-1的生物活性通过刺激石斑鱼胚胎细胞系（GP细胞系）和MFC-7细胞增殖的能力得到证实。目前的结果表明，巴斯德毕赤酵母表达系统可用于生产用于研究和水产养殖应用的功能性rgIGF-1。
