重组甘精胰岛素的可溶性表达研究-东南大学学报医学版

重组甘精胰岛素的可溶性表达研究

单位：南京大学医药生物技术国家重点实验室, 江苏南京 210093

分类号：Q578;Q786

出版年·卷·期（页码）：2013·32·第六期（663-669）

摘要：

目的：构建甘精胰岛素（ING）表达载体，提高ING在大肠杆菌中的可溶性表达。方法：在传统方法中ING的A、B链之间用C肽连接，本实验在ING的A、B链之间插入大肠杆菌硫氧还蛋白质（Trx）来替代C肽，进行ING的融合表达（ING-Trx），并进一步使用多分子伴侣GroEL、GroES、触发因子进行共表达。结果：可溶性的ING-Trx融合蛋白约占该目的蛋白质总表达量的35%；多分子伴侣共表达后可溶性ING-Trx的产量提高到ING-Trx总蛋白的78%，初步纯化后其产量为4.5 mg·L^-1，是使用分子伴侣前的3倍。结论：用Trx代替C肽不影响A、B链的有效折叠，采用分子伴侣共表达可以显著提高目的蛋白的可溶性。

Objective: To construct expression vector of insulin glargine(ING) and increase soluble expression of ING. Methods: C peptide was used to join A chain and B chain of ING in previous studies. In the present research, thioredoxin (Trx) was inserted between A chain and B chain of ING to replace C peptide. Co-expression of molecular chaperones GroEL, GroES, trigger factor and ING was also used to promote folding of recombinant ING-Trx protein. Results: Soluble ING-Trx accounts for 35% of total expressed ING-Trx while soluble ING-Trx increased to 78% of total ING-Trx protein after co-expression. The yield was about 4.5 mg·L^-1 after preliminary purification, three times as much as the production without co-expression of molecular chaperones. Conclusion: In summary, A chain and B chain can fold properly when Trx is used to replace C peptide. Co-expression of ING-Trx with molecular chaperones can greatly increase soluble expression of insulin glargine.

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