论文部分内容阅读
胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。
Proinsulin (Pins) is a precursor of insulin synthesis. In E. coli expression system, which usually exists in the form of inclusion bodies, need to go through the denaturation refolding and other subsequent processing in order to get active insulin. Cell-free protein synthesis (CFPS), as a novel in vitro protein synthesis method, breaks through the physiological limitations of cells and has been successfully used in the production of a variety of recombinant protein drugs. In order to explore a new method of insulin synthesis to meet its needs in the development of new routes of administration, this study used the CFPS system for the soluble expression of proinsulin. The proinsulin fusion protein was successfully expressed in the CFPS system by increasing its solubility by fusion of proinsulin with the fluorescent protein. Finally, Pins-mCherry fused with red fluorescent protein was identified by Western blotting. Quantitative analysis of Pins-eGFP fusion protein in supernatant was performed by using microplate reader. The results showed that Pins -eGFP was partially soluble, its expression level was (12.28 ± 3.45) μg / m L. This study, for the first time, resulted in the soluble expression of fusion proinsulin in the CFPS system. The strategy of fusion of the fluorescent proteins significantly enhanced the solubility of proinsulin, which laid the foundation for exploring new methods of insulin synthesis and developing new insulin delivery methods based on the CFPS system The foundation.