Cloning, Expression, Purification And Biological Activity Study Of GIP Analogs

Glucose-dependent insulinotropic peptide, known as gastric inhibitory polypeptide in the past, is an incretin and potentiates insulin secretion in pancreaticβ-cells in the presence of glucose. Native GIP has little pharmaceutical application because of its short life time in vivo, which is about 5-7 minutes. There are two major problems made GIP's short life time in vivo:GIP undergoes rapid proteolytic cleavage by the ubiquitous serine protease, dipeptidyl peptidaseⅣ(DPPⅣ) at their alanine residue(Ala2), producing N-terminally truncated peptides incapable of stimulating insulin secretion; and the primary route for GIP clearance appears to be the kidney. To obtain long life time GIP, site-directed mutation was conducted by PCR, and the corresponding GIP mutants (analogs) were got through E.coli expression system and then their biological activities were studied in animal model.Ala2 of GIP was substituted by Ser2 to generate DPPIV resistant GIP analog, named mGIP2. It can escape DPPIV digestion and remain the insulinotropic activity. Another GIP analog has the same sequences of mGIP2 and has an additional cysteine at the C-terminal, named mGIP243. Native GIP gene was obtained by PCR from mGIP2. All the three genes were cloned to E.coli expression vector pET32a(+) and were named pET32a-mGIP2, pET32a-mGIP243 and pET32a-nGIP correspondingly. Those recombinant plasmids were transformed to expression host E.coli BL21(DE3) to get relevant recombinant E.coil strains.Culturing and expression conditions of recombinant GIP analog strains were optimized as follow:4%(v/v) seed bacteria was inoculated and let culture grow to OD600 around 0.6-0.8; then a final concentration of lmmol/L IPTG was added and 4 hours more culturing at 37℃,210r/min was performed. Bacteria was collected and resuspended. Most unexpected proteins were eluted in IDA80, and target fusion protein was eluted in IDA 100 and IDA200. Fusion protein was further digested by EK proteomes at 25℃for 16 hours. The corresponding mixture was loaded to a second affinity chromatography and fraction of IDA20 elution was collected to get the native GIP and its analogs mGIP2 and mGIP243. Animal assay showed that comparing to nGIP, mGIP2 and mGIP243 had better activity in vivo. The in vivo insulinotropic effect was less than 30 minutes for nGIP, and 30 minutes and 90 minutes for mGIP2 and mGIP243 respectively. mGIP243 had even longer and better activity than mGIP2, which suggested that mGIP243 not only was resistant to DPPⅣ, but also escaped the glomerular filtration through binding to albumin. This hypothesis was proved by western blot. mGIP243 conjugated to albumin automatically in vitro, which suggested that it may have the same reaction in vivo. In conclusion, mGIP243 is a potential drug candidate for treating diabetes mellitus.There is a resistance to the insulinotropic action of native GIP in type 2 diabetic patients and GIP make fat accumulating in type 2 diabetic patients. These aspects limit the application of GIP. Recent research found that substitution of Glu3 of GIP with Pro3 provides antagonist effects. Interestingly, pro3GIP can prove metabolism disorder in obesity-diabetic and high-fat diets animal model.In this study, Glu3 of native GIP was substituted by Pro3 to obtain GIP receptor (GIPR) antagonist; further more, an additional cysteine was added to C-terminal to get another GIPR antagonist. These GIP analogs were named pro3GIP and pro3GIPC respectively. The genes were synthesized by PCR based on mGIP2 and mGIP243 genes and were cloned into pET32a(+). The corresponding recombinant strains were named E.coli BL21(DE3)/pET32a- pro3GIP and E.coli BL21(DE3)/pET32a-pro3GIPC. Expression and purification conditions were optimized and were similar as described above.In vivo study shown that pro3GIPC was a competitive antagonist of GIPR, and had a higher binding effect when in high concentration. At a molar ratio of 4:1, pro3GIPC completely inhibited the insulinotropic effects of mGIP2. All results showed that pro3GIPC was a new GIPR antagonist, which had the same characteristics of pro3GIP.After 11 days administration of pro3GIP or pro3GIPC, obesity diabetic mice db/db mice had improved non-fasting plasma glucose. The glucose lowering effect of pro3GIPC was better than that of pro3GIP, suggesting that pro3GIPC might have longer life time in vivo. The glucose lowering effect vanished after stopping administration, showed the glucose lowering effect of pro3GIPC is reversible. Glucose tolerance, glycated hemoglobin and other metabolic impairs of the pro3GIPC treated mice was improved after 11 days treatment. Insulin sensitivity tested by IPGTT on obesity diabetic mice showed that mice treated with pro3GIPC had improvement compared with untreated group. The group treated with pro3GIPC also had better glycated hemoglobin improvement and lower triglyceride. In a word, pro3GIPC improved metabolic disorders through blocking GIPR signaling. By adding a cysteine to pro3GIP, the new GIP analog pro3GIPC was a better GIPR antagonist compared to pro3GIP and could be a new way for T2DM treatment.