重组甘精胰岛素的可溶性表达研究

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4．5mg·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。

Study of soluble expression of recombinant insulin glargine

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 chaoerones can greatly increase soluble exnression of insulin glargine.