Cloning, Expression And Biological Activity Analysis Of Glucagon-like Peptide-1 Mutant Gene

Glucagon-like peptide-1(GLP-1) is a physiological glucose-dependentincretin, secreted by intestinal L cells. GLP-1 has two active forms, GLP-1(7-36)amide and GLP-1(7-37). GLP-1 is released in blood rapidly inresponse that L cells are activated after meal ingestion and oral or intravenousinjection glucose. Because GLP-1 has an important role in regulatingpostprandial blood glucose concentrations, there is currently great interest intrying to develop GLP-1 as a new therapeutic agent for diabetes.We have cloned and purified recombinant GLP-1. In this study, we willanalyse biological activity of recombinant GLP-1, clone and express GLP-1mutant gene, purify recombinant GLP-1 mutant protein, and study itsbiological activity. The research results could be used to producerecombinant GLP-1 mutant on a scale using gene engineering techniques andprovide data for clinical treatment of diabetes in the future. This subject studyinvolved four contents: (1) biological activity analysis of recombinantGLP-1(7-36)NH₂ and GLP-1(7-37) in vivo; (2) cloning and expression ofGLP-1(7-37) mutant gene; (3) purification of GLP-1(7-37) mutant protein; (4)biological activity analysis of GLP-1(7-37) mutant protein.Methods:1. SD rat model with hyperglycemia was developed by intraperitonealinjection in these normal SD rats with 50% glucose 18mmol/kg.Recombinant GLP-1(7-36)NH₂ and GLP-1(7-37) were administered atdifferent doses (2μg/100g, 4μg/100g and 8μg/100g), then blood samples werecollected and the levels of plasma glucose and insulin were determined.Subsequently, the experimental 1 model diabetic rats were produced withstreptozotocin on 60mg/kg. Recombinant GLP-1 (7-36)NH₂ (11.2pmol/kg·min) was intravenously injected into the diabetic rats for 120 minand the levels of plasma glucose were measured.2. Using recombinant GLP-1(7-37) expression system, the 2nd alaninewas changed to be glycine by means of site-directed mutagenesis. The mutant gene was amplified by PCR, and cloned to the plasmid vector pGEM-7Z(+)after appropriate cleavage with restriction endonuclease and then insertedinto the MCS of the fusion expression vector pGEX-4T-3. The recombinantplasmid was transformed into E. coli. BL21(DE3) and induced by IPTG.3. The fusion protein was prepared from sonication of bacteria byglutathione-Sepharose 4B affinity chromatography. Following cleavage offusion protein by cyanogen bromide, the recombinant mutant²Gly-GLP-1(7-37) was released from fusion protein, and purified usingSephadex G-25, QAE-Sepharose FF and RP-C₁₈ column chromatography.Then the purified recombinant mutant ²Gly-GLP-1(7-37) was analysed byWestern blotting.4. The recombinant GLP-1(7-37) and ²Gly-GLP-1(7-37) were incubatedwith dipeptidyl peptidase (DPP)â…£at 37℃, and the activity of resistance toDPPâ…£degradation was analysed by HPLC. Effects of recombinantGLP-1(7-37) and ²Gly-GLP-1(7-37) on plasma glucose and insulin wereexamined in SD rats by using the method of intraperitoneal injection of eitherpeptide (4μg/100g).Results and discussion:1. Compared with glucose group, plasma glucose levels weresignificantly decreased after administration 4μg/100g, 8μg/100g recombinantGLP-1(7-36)NH₂ and GLP-1(7-37) in the experimental SD rat model.Besides, the levels of insulin in plasma were also apparently increased inthese rats as compared with glucose rats (P＜0.001). Both GLP-1(7-36)NH₂and GLP-1(7-37) could improve the levels of plasma glucose and insulin, butthese indexes showed no significant difference between them. In the type 1diabetic rats, plasma glucose concentrations were (17.04±1.31)mmol/L(P＜0.05) after 60 min by intravenous injection GLP-1(7-36)NH₂, and glucoseconcentrations were (11.98±1.05)mmol/L(P＜0.001) after 120 min. GLP-1treatment could improve plasma insulin levels in the experimental diabeticrats model. 2. The recombinant plasmid DNA was digested with BamHâ… and Xhoâ… and GLP-1 gene mutation was testified by DNA sequencing. The resultsdemonstrated that the mutated ²Gly-GLP-1(7-37) was successfully insertedinto the pGEX-4T-3 vector. SDS-PAGE and scan analysis showed the fusionprotein GST-²Gly-GLP-1(7-37) was expressed in a soluble form andamounted to 23.5% of the total bacterial protein.3. The purity of ²Gly-GLP-1(7-37) was more than 98% through affinitychromatography, QAE-Sepharose FF anion exchange chromatography andRP-C₁₈ reverse chromatography. Western blotting analysis confirmed that²Gly-GLP-1(7-37) could be specificly recognized by the antibody ofGLP-1(7-37).4. Incubated with DPPâ…£at 37℃after 4 h, above 90% of GLP-1(7-37)was cleaved by DPPâ…£, whereas, about 85% of ²Gly-GLP-1(7-37) wasresistant to DPPâ…£degradation, and remained intact. Plasma glucoseconcentrations were significantly lower and insulin concentrations higherafter intraperitoneal injection of both peptides than glucose alone. Moreimportantly, individual glucose values at 15 and 30 min were significantlylower after administration of ²Gly-GLP-1(7-37) than GLP-1(7-37)(P＜0.05).This was associated with a significantly higher insulin response afterinjection of ²Gly-GLP-1(7-37) than GLP-1(7-37)(P＜0.05). These resultssuggested that NH₂-terminally modified recombinant ²Gly-GLP-1(7-37)mutant may be useful in the treatment of type 2 diabetes mellitus.