重组人胰高血糖素样肽-1突变体的分离纯化及活性分析

对基因工程构建的含人胰高血糖素样肽1(hGLP1)突变体的工程菌株进行诱导表达,分离纯化N末端第二位突变的2GlyhGLP1突变体.IPTG诱导4h,收获的菌体经超声破碎后,裂解液用GlutathioneSepharose4B亲和层析纯化GST2GlyhGLP1融合蛋白,经CNBr裂解、SephadexG25柱脱盐、QAESepharoseFF阴离子交换柱层析和RPC18柱脱盐,得到纯度大于98%的重组2GlyhGLP1.Western印迹分析证实,该突变体可被特异性hGLP1抗体所识别.生物学活性分析表明,2GlyhGLP1具有明显的降血糖活性和促胰岛素分泌活性(P<0.001).

Purification and Activity Analysis of Recombinant Human Glucagon-like Peptide-1 Mutant

To purify recombinant human glucagon-like peptide-1 mutant ( 2Gly-hGLP-1) and study its biological activity from fusion protein GST- 2Gly-hGLP-1 expressed in E. coli BL21 (DE3)/pGEX- 2Gly-hGLP-1, GST- 2Gly-hGLP-1 fusion protein was prepared from sonication of bacteria by glutathione-Sepharose 4B affinity chromatography. Following cleavage of GST- 2Gly-hGLP-1 by cyanogen bromide, the recombinant mutant 2Gly-hGLP-1 was released from fusion protein, and purified using Sephadex G-25, QAE-Sepharose F F and RP-C_ 18 column chromatography. The purity of recombinant 2Gly-hGLP-1 was more than 98 % by HPLC analysis. After purification, Western blotting analysis of 2Gly-hGLP-1 confirmed that it could be specially recognized by the antibody of hGLP-1. The biological activity was examined by intraperitoneal injection of 2Gly-hGLP-1 into rat,as compared with glucose-injection control group,plasma glucose concentrations were markedly lower (P<0.001) while insulin levels higher 15,30 and 60 min after concomitant injection of 2Gly-hGLP-1 plus glucose(P<0.05).These data demonstrated that 2Gly-hGLP-I mutant had the similar effect as the native one.