无细胞蛋白合成体系实现胰岛素原可溶性表达

Synthesis of soluble proinsulin in a cell-free protein synthesis system

摘要:

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/mL。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。

Abstract:

Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.