Expressing Of ~2Gly-hGLP-1-HSA Fusion Protein In Pichia Pastoris

The incretin hormone glucagon-like peptide-1(GLP-1)is capable of ameliorating glucose-dependent insulin secretion in subjects with diabetes.It stimulates endogenous insulin secretion in a glucose-dependent manner,while at the same time it also decreases blood glucagon levels and reduces gastric emptying by slowing gastric motility.The combined effect of these mechanisms makes GLP-1 a potent blood glucose-lowering agent.The blood glucose-lowering effect,as well as the glucose dependency(ie,stimulation of insulin secretion only when plasma glucose levels are above normal)of this incretin hormone makes it an interesting candidate for the treatment of type 2 diabetes.However,native GLP-1 has a short circulating half-life(t_1/2≈2 min)that results mainly from rapid enzymatic inactivation by dipeptidyl-peptidaseâ…£(DPP-â…£),and/or renal clearance.Therefore, continuous subcutaneous infusion by pump is necessary to maintain GLP-1 action in vivo.DPP-â…£-resistant GLP-1 analogues,and other formulations,appear to be promising therapeutic drug candidates for the treatment of diabetes,but these peptides require once- or twice-daily injections and/or combination therapies with oral diabetic medications.The E.coli and the mammalian cell expression system have obvious limitation.The purpose of this work was to gain high-level expression from secretive expression of eukaryote.We used the Pichia pastoris expression system,because it is very mature.The yeast transforments were screened to obtain high expression strains by the test and analysis of expressed supernatant of shake-flask culture in 10%SDS-PAGE.We acquired the recombinant protein from the supernatant,and explored the purification condition for the expressed protein,at the same time we identified purified protein for its bioactivity.The work made a progress on the structure,function and industrialization reseach.In this study,we previously synthesized the nucleic acid of ²Gly-hGLP-1,which is an analog of hGLP-1 with Ala substituted by Gly at position 2,according to the prefered codon usage of Pichia pastoris.C-terminal of ²Gly-hGLP-1 and N-terminal of HSA was linked through a five-Gly spacer. The gene encoding ²Gly-hGLP-1-HSA fusion protein was obtained by chemical synthesis and overlap extension PCR.Then we constructed the pPIC9/²Gly-hGLP-1-HSA vectors and transformed into Pichia pastoris strains by electroporation.Through phenotype selection and expression assay,the expressing strain was obtained.After cultivation in 5 L bioreactor and purification,the purity of recombinant ²Gly-hGLP-1-HSA exceeded 95%.IPGTT(i.p.glucose tolerance test) revealed that the ²Gly-hGLP-1-HSA expressed in Pichia pastoris can lower plasma glucose in vivo. The main results were as follows:1.We obtained the ²Gly-hGLP-1 by artificial synthesise and polymerase chain reaction (PCR),gained the reactions of the PCR were carried out for 2 cycles at 94℃for 5min,,65℃for 5min,72℃for 10min.single-step assembly the gene of ²Gly-hGLP-1 for 90bp length by PCR.2.We obtained the ²Gly-hGLP-1-HSA by fusing ²Gly-hGLP-1 gene and HSA gene,gained the reactions of the PCR were carried out for 30 cycles at 94℃for 30s,50℃for 30s,72℃for 2min,extension at 72℃for 10min.The ²Gly-hGLP-1-HSA gene was 1884bp length.3.The ²Gly-hGLP-1-HSA gene was digested by EcoRI+XhoI.The digested products were cloned into expression vector pPIC9,and the recombinant plasmids were identified by restriction endonuclease enzyme analysis and 1%agarose gel electrophoresis.4.We prepared the strains of P.pastoris(GS115),the recombinant plasmids (pPIC9/²Gly-hGLP-1-HSA)transformed into the strains by electroporation,and screened the positive transformant were identified by MD plate and the electroporation parameter was voltage:2.0Kv,time:4.5ms.5.We gained the starting induced time and the ending time:cultured the engineering strain into the medium,when OD600=1,then put the 0.5%methanol into the medium,and put 0.5% methanol every 12 hours,we got the purpose protein strips by checking the 10%SDS-PAGE electrophoresis(²Gly-hGLP-1-HSA protein was 69.5KD),and also we got the high-level expression purpose protein by 84 hours inducement.6.After explored the purification condition,the fusion protein can stably expressed in the 5L fermentor.The expression level exceeded 100mg/L.7.The purification technics of the ²Gly-hGLP-1-HSA protein were obtained.After SP Sepharose F.F,P.henyl Sepharose HP,Source 30 Q and Superdex 75,the purity of recombinant ²Gly-hGLP-1-HSA exceeded 95%.8.The biological activity of the ²Gly-hGLP-1-HSA protein was examined by KM rats,as compared with saline-injection control group,plasma glucose concentrations were lower(P＜0.05).These data demonstrated that ²Gly-hGLP-1-HSA can reduce the inappropriate rise in glucose.