Production And Biological Activities Of A Novel Recombinant Human Glucagon-like Peptide-2

Glucagon-like peptide-2(GLP-2) is a gut-specific nutritional factor which is one of the proglucagon-derived peptides (PGDPs). GLP-2is a33amino acid peptide with the sequence HADGSFSDEMNTIDNLAARDFINWLIQTKITD and its molecular weight is about3.9kDa. GLP-2has various biological activities in intestinal including stimulating the mucosal growth of small and large intestines, inhibiting of enterocyte and crypt cell apoptosis, accelerating glucose transport and GLUT-2expression, promoting nutrient absorption, inhibiting of gastric emptying and gastric acid secretion, reducing intestinal permeability, stimulation of intestinal blood flow and relaxing intestinal smooth muscle. In view of the above biological activities, GLP-2was developed for the treatment of short bowel syndrome, inflammatory bowel disease and radiation-induced enteritis and other bowel diseases. However, the biological half-life of circulating GLP-2is very short (about7minutes in humans) due to its rapid degradation by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV) and extensive renal clearance. If the second amino acid residue alanine was substituted by glycine, it can resistant to DPP-IV mediated degradation, leading to extended half-life and enhanced biological activity.Although GLP-2is likely to be broadly applied in many clinical and experimental trials, but the high costs limited its clinical use. At present, most of the GLP-2and its analogues used in clinical and experimental trials are produced by chemical synthesis method which not only the technology is both complex and costly. In addition, because of its lower molecular weight, it’s unlikely to be expressed in (E. coli) and prepared in large-scale by genetic engineering. For this reason, the aim of our research is to acquire GLP-2or its analogues by a more convenient and ecnomical method.In our research, the gene encoding the concatemer GLP-2②was designed and synthesized and then cloned into pET-22b prokaryotic expression vector, the correct recombinant plasmid pET-22b(+)-GLP-2②was transformed into (E. coli) strain BL21(DE3). The recombinant (E. coli) could express the expected concatemer GLP-2②when induced by β-isopropylthio-galactoside (IPTG). The expressed GLP-2②could be purified by salting-out and anion exchange chomatography and the purity of GLP-2②was over95%as detected by size-exclusion chomatography HPLC. After confirming the authority of the recombinant GLP-2②by Western-blot and Mass spectrometry, the activity of GLP-2②was further investigated and our data showed that GLP-2②could promote the proliferation of Caco-2cell line in virto and could significantly increase the villus height and intestinal cell proliferation of Balb/c mice in vivo. In addition, the biological activity of recombinant GLP-2②is higher than that of the chemically synthesized GLP-2monomer.In conclusion, we got a more simple and economical method to express and prepare active GLP-2②, which laid a foundation for the use of GLP-2and its analogues in short bowel syndrome, radiation-induced enteritis and other bowel diseases.