降糖肽Aglycin的高效表达及活性鉴定

High-level Expression and Activity Determination of Hypoglycemic Peptide Aglycin

基于自组装肽ELK16能在大肠杆菌细胞内自聚集的特性,本研究将Aglycin通过一个内含肽Mxe GyrA与自组装肽连接,构建能在体外自剪切的重组表达载体pET30a-Aglycin-Mxe-PT-ELK16。将重组质粒转化大肠杆菌BL21(DE3)并进行蛋白表达条件的优化,得到目的蛋白聚集体,随后对聚集体进行DTT切割条件的优化,经过进一步纯化最终可得到纯度为98.15%、产量为5.53mg/g菌体湿重的Aglycin肽。采用基因工程手段重组表达Aglycin的产量比目前化学提取方法提高了近100倍。此外,经体外实验证明,Aglycin对α-葡萄糖苷酶抑制的IC50(36.48μmol/L)比阿卡波糖(991.33μmol/L)低,说明Aglycin比阿卡波糖有更强的α-葡萄糖苷酶抑制活性,这进一步证明Aglycin有开发为α-葡萄糖苷酶抑制剂的潜力。

In this study, based on the self-aggregation characteristics of the self-assembling ability of ELK16 in Escherichia coli, Aglycin was linked to the self-assembling peptide via an intein, Mxe Gyr A, to construct a recombinant expression vector p ET30a-Aglycin-Mxe-PT-ELK16 capable of self-cleavage in vitro. The recombinant plasmid was transformed into E. coli BL21(DE3), and the protein expression conditions were optimized to obtain the target protein aggregates. Then, the DTT cleavage conditions were optimized for the aggregates. After further purification, Aglycin peptide with a purity of 98.15% and yield of 5.53 mg/g cell pellet wet weight was finally obtained. The yield of Aglycin peptide obtained by recombinant expression was almost 100 times higher than that of the currently used chemical extraction method. In addition, in vitro experiments have shown that the IC50 value(36.48 μmol/L) of Aglycin peptide for α-glucosidase inhibition was lower than that of acarbose(991.33 μmol/L), indicating that the inhibitory activity of Aglycin peptide against α-glucosidase was much stronger than that of acarbose. This further demonstrates the potential of Aglycin peptide as an α-glucosidase inhibitor.