Ma Sanyuan, Xia Xiaojuan, Li Yufeng, Sun Le, Liu Yue, Liu Yuanyuan, Wang Xiaogang, Shi Run, Chang Jiasong, Zhao Ping, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, People's Republic of China.
Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, 2, Tiansheng Road, Beibei, Chongqing, 400716, China.
Mol Genet Genomics. 2017 Aug;292(4):823-831. doi: 10.1007/s00438-017-1311-7. Epub 2017 Mar 29.
Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.
为满足对重组蛋白日益增长的需求，人们开发了各种转基因生物反应器系统。家蚕的丝腺具有成为用于商业规模生产重组蛋白的经济高效生物反应器的巨大潜力。然而，从当前丝腺表达系统获得的蛋白质实际产量过低，无法大规模溶解和纯化这些蛋白质。在此，我们提出一种策略，即减少内源性丝胶蛋白将提高外源蛋白的表达产量。利用转基因RNA干扰技术，我们成功将BmSer1的表达降低至50%。总共建立了26个在BmSer1启动子控制下在中部丝腺（MSG）中表达盘基网柄菌红色荧光蛋白（DsRed）的转基因品系，以分析重组体的表达情况。qRT-PCR和蛋白质印迹分析表明，在BmSer1敲低品系中，DsRed在mRNA和蛋白质水平上的表达均显著增加。我们还对茧中DsRed/BmSer1的分布以及DsRed蛋白积累对丝纤维形成过程的影响进行了分析。本研究不仅描述了一种提高MSG生物反应器中重组蛋白表达的新方法，还提出了一种优化其他生物反应器系统的策略。
