Expression And Purification Of Human Globular Adiponection Glucagen-like Peptide-1 Analogues Fusion Protein Including Enterokinase Site

Insulin resistance and insulin excreting deficiency are the primary pathological and physiological changes of type 2 diabetes. Interaction between these changes leads to the appearance and development of type 2 diabetes. At present, there are no medicine aiming at both insulin resistance and insulin excreting deficiency to treat this disease. Adiponetin is a protein excreted by lipocyte particularly. Adiponcetin correlates with insulin resistance. It can increase insulin sensitivity of peripheral tissue, decrease blood glucose and blood lipid. Beside this, adiponectin also has an protective action against inflammation and atherosclerosis. Its functional domain is globular C-terminus, with the same function as full adiponectin. In skeletal muscle, globular adiponectin (gAdiponectin) has higher activity than full adiponectin. Glucagon-like peptide-1 (GLP-l) is a kind of hormone secreted by islet a cell and intestinal L cell. Its N-terminal is functional, which can promote insulin synthesis and release by binding with GLP-l receptor in a glucose-dependent insulinotropic manner. Its half-life in vivo is very short. GLP-l is cleared by dipeptidyl peptidase-rV(DPP-IV)and kidney ,which limit its clinic appliance greatly. So it is important to find some GLP-l analogues(GLP-l-A) which can increase resistance to DPP-IV to prolong metabolic stability.By recombinant technique of gene, this study connected two genes coding globular adiponectin and GLP-l-A respectively to construct prokaryotic expressionvector PET28a-gAdiponectin-GLP-l-A. After the vector transfected prokaryotic expression bacteria, the human gAdiponectin-GLP-l-A fusion protein including enterokinase site was expressed and purified, which lays the foundation for enterokinase cleavage and studying the activity of the human gAdiponectin -GLP-l-A fusion protein, to gain an new protein which can amelioration not only insulin resistance but also insulin excreting.Materials and methods 1 Construction of the recombinant vector PET28a-gAdiponectin--GLP-l-APrimers encoding gAdiponectin(P3 P4) and GLP-1 -A(P1 P2) were designed in accordance with prokaryotic expression vector PET28a and gene announced in GenBank. Genomic DNA was extracted from human blood cell. GLP-l-A was gained by PCR, then inserted into PET28a to create PET28a -GLP-l-A. The resulting construct, PET28a-GLP-l-A, was confirmed by restriction mapping and sequencing. Total RNA was isolated from human adipose tissue. GAdiponectin was amplified by RT-PCR, then inserted into PET28a-GLP-l-A to produce PET28a-gAdiponectin-GLP-l-A, which was confirmed by another two restriction enzyme and sequencing.2 Expression and purification of the human gAdiponectin-GLP-l-A fusion protein including enterokinase siteThe constructed recombinant vector PET28a -gAdiponectin -GLP-l-A was transfected into prokaryotic expression bacteria BL21(DE3). Isopropylthio-P-D-galactoside(IPTG) was added to cultured bacterium to generate the fusion protein. The expressed fusion protein was verified by 12% SDS-PAGE and western blot. After enlarging the expression system, nickel resin was used to purify the fusion protein. The purified fusion protein including enterokinase site was confirmed by 12 % SDS-PAGE and western blot.Result1 The 426bp gAdiponectin and the 159bp GLP-l-A were showed in 1.5% agarose gel electrophoresis as expected. PET28a-gAdiponectin-GLP-l-A was digested with BamH I /Hind III and Xba I /Hind III . The enzymed fragment included 426bp and 683bp respectively, which indicated that the fusion protein gene was inserted into prokaryotic expression vector of PET28a successfully. DNA sequence analysis showed that inserted direction and sequence are completely accurate.2 12% SDS-PAGE analysis revealed that the fusion protein was induced , withabout expected molecular weight of 25KD. Western blot analysis with His monoclonal antibody demonstrated that the fusion protein exited in both supernatant and sediment, but mainly in the latter. High purity of the fusion protein gAdiponectin-GLP-l-A including enterokinase site was obtaine...