Preparation Of Recombinant Human Glucagon-like Peptide-1 By Gene Engineering And Studies On Its Biological Activities

Glucagon like peptide-1 is a gastrointestinal and incretion hormone released from the enteroglucagon cell (L-cell) in the small intestine of human during input of glucose from food or injection. Since GLP-1 was discovered in 1980s, its physiological features, including stimulation insulin secretion and gastrointestinal peristalsis, have demonstrated by experiments in vitro and vivo in many laboratories. Several structural forms of GLP-1 have been found in the human body; however, two of them, GLP-1(7-36)NH2 and GLP-1(7-37), are the main active forms.GLP-1 plays an important role in regulation of postprandial insulin release from the ?cell of pancreas. In the patients with type II diabetes, exogenous GLP-1 could reduce postprandial plasma glucose elevations by stimulating insulin release and inhibiting glucagon secretion. Therefore, GLP-1 is considered a great potential reagent for pharmacotherapy of type II diabetes in the clinic.The purpose of this research project was to establish techniques for preparation of recombinant GLP-1 and to study the biological activities of recombinant GLP-1 in rat models in vivo. The research results could be used to produce rhGLP-1 on a larger scale using geng engineering techniques of recombinant GLP-1 for clinical treatment of type II diabetes in the future.Considering the different characteristic of GLP-1(7-36)NH2 and GLP-1 (7-37) in the human body, the preparation procedure for recombinant both were studied.This research describes: (1) cloning of hGLP-1, constructing ofhGLP-1 expression vectors and engineering bacterial strains expressing hGLP-1; (2) developing a fermentation system to grow bacteria at a high density and with high expression of rhGLP-1 ; (3) separation and purification of rhGLP-1 and prerhGLP-1; (4) amidation of prerhGLP-1 in vitro; (5) assessment of biological activities of rhGLP-1 in rat models in vivo.Methods:1. Six oligonucleotides fragments were synthesized according the genetic codon of prehGLP-1(7-36)NH2 and hGLP-1(7-37) cDNA. Two hGLP-1 cDNAs were constructed by the procedure of annealing and ligation with oligonucleotides fragments. The recombinant plasmids of hGLP-1 cDNA fused with GST gene were transformed into E. coli BL21(DE3) and expression of GST-rhGLP-1 was significantly induced by IPTG.2. The optimization of induction conditions were carried out in 500 ml shake flasks, followed by using a 5 L fermentor with high density culture to produce GST-rhGLP-1 in E. coli BL21(DE3)/pGEX-hGLP~l.3. The fusion proteins were purified from lysates with glutathione Sepharose 4B affinity chromatography. The prerhGLP~l(7-36)NH2 and rhGLP-1 (7-37) were obtained by a series of purification steps comprised of CNBr cleavage, QAE sepharose FF and HPLC purification.4. The C-terminal amidation of prerhGLP~l(7-36)NH2 was performed by transacylating with CPD-Y.5. The assessment of the biological activities of rh.GLP-1 (7-37) and rhGLP-1(7-36)NH2 was performed by using hyperglycemic SD rats and a diabetic rat model in vivo.Results and Disscussions:1. The construction of two recombinant plasmid DNAs was confirmed by digestion with restrictive endonuclease BamH I and Xho I , followed by DNA sequencing. The results indicated that prehGLP-1(7-36)NH2 and hGLP-15(7-37)cDNA were inserted into the pGEX-4T~3 vector and that the fusion proteins GST-rhGLP-1 were expressed by the transformed bacterial, as shown in SDS-PAGE gel.2. Batch and fed-batch culture were used to obtain high density culture cell by controlling carbon-nitrogen concentrations and keeping oxygen at 30%~40%. The weight of harvested cells could reach to 150g/L; the final density of grown bacterial was 52 0. D6oo; the products of fusion protein GST-rhGLP-1 was 2. Ig/L.3. The purities of prerhGLP-1(7-36)NH2 and rhGLP-1(7-37) were over 85% respectively by HPLC analysis. ESI-MS showed that the molecular weight of prerhGLP-l(7-36)NH2 and rhGLP-1 (7-37)were as expected.4. The prerhGLP-l(7-36)NH2 was transacylated by CPD-Y. The purity of resulting rhGLP-1(7~36)...