NGLP-1R's Expression And Purification In A Prokaryote Expression System

Glucagon-like peptide-1 (GLP-1) is a hormone that engages GLP-1 receptor , It is mainly produced in enteroendocrine L cells of the gut and is secreted into the blood stream when food containing fat, protein hydrolysate and/or glucose enters the duodenum. It is known to increase insulin secretion and decrease plasma glucose levels in type 2 diabetes, now it is being used to treat type 2 diabetes as a type of injection. GLP-1 can also slow down gastrointestinal motility, so it has been regarded as a useful body weight controller.GLP-1 engages a specific G-protein coupled receptor GLP-1R. GLP-1R is a specific seven-transmembrane receptor guanine nucleotide-binding protein (Gprotein) coupled receptor (GPCR). The rat and human GLP-1Rs exhibit a 95% amino acid homology and are 90% identical, differing at 42 amino acid positions, there has been only one functionally distinct GLP-1R described. The human GLP-1R gene is located on the long arm of chromosome 6p21. GLP-1R is a member of the Class B family , within Class B the receptors for the peptide hormones form a subclass of the glucagon receptor family. An amino-terminus extra-cellular domain of 100-150 amino acids characterizes the structure of Class B peptide receptors. There are six highly conserved cysteine residues in the extracellular region that have been composed to three disulphide bonds, these bonds can sustain the N-terminal in to a globosity domain. GLP-1R's mRNA has been found in rodent's pancreas, brain, kidney, lung, heart, major blood vessels and so on. In intestine the distributing of GLP-1R is increased from the end of the jejunum to the colon.The prevailing ligand binding model of family B 7TM GPCRs suggests that GLP-1 and its receptors ligand can be regarded as a two-domain interaction mechanism.First, the Nt-domain of the receptor interacts with the C-terminal segment of the ligand, and this interaction is a major determinant of selectivity between the different homologous peptide ligands.Second, the N-terminus of the ligand interacts with the extracellular surface of the receptor core-domain (the 7TM helices and connecting loops), which leads to receptor activation. In fact, an 11 residue mimetic of the GLP-1 N-terminus is sufficient to activate GLP-1R.In this study we used a prokaryote expression system to produce a kind of activated GLP-1 N-terminus (nGLP-1R) (E24-Y145). The production can be used as the combinator of screening the GLP-1mimetics from peptide libraries and as a of the ligand capability between the receptor and the GLP-1 mimetics.The aim gene was purified from RINm-5F cell. First use the RNA as the template of RT-PCR, then use the product of RT-PCR as the template of the nest PCR. The product of the second PCR was examined by 2% agarose electrophoresis. The result of the electrophoresis the length of the DNA segment was 392bp, it approved that the aim DNA segment was cloned correctly.Extracted DNA segment from agarose gel, liganded it into pTG19-1T vector, the clone vector was transformated into competent JM109 E coil. cell to produce more clone vector. Then the clone vector was digested with restriction endonuclease EcoRI and HindIII. The results was presented by gel electrophoresis. The result showed that the segment cut by the restriction endonuclease has a length of 392bp, it approved that the clone vector has contributed correctly.The segment cut from the clone vector has been liganded into pET32a plasmid as the expression vector, pET32a has a Trx gene, a His-tag and a enterokinase cutting site. Digest the clone vector and pET32a, then extracted the aim segment, liganded with pET32a, the expression vector has been examined by gel electrophoresis,. The results illuminated that the expression vector has contributed correctly.The expression vector was transformated into the competent BL21(DE3) E coil.. Used IPTG as a revulsant. Then used SDS-PAGE electrophoresis to checkup the yield of the fusion protein. The result show that there're a 33.5kd striation, the same as the fusion protein's weight. So it approved that the prokaryote expression system has been built correctly.Expression the fusion protein, harvest by centrifugation and sonication. The SDS-PAGE electrophoresis exhibited that most of the fusion protein was in the supernatant.The supernatant has been purification by the Ni-NTA immobilized-mental affinity chromatography, and then digested by enterokinase. The remnant of the fusion protein is the Trx-His tag and the aim protein.The remnant was purificated by the Ni column, the Trx has his-tag, and ligand with Ni2+, so there are only the aim protein remained in the remnant. The SDS-PAGE electrophoresis had had confirmed it.In this study, we used a prokaryote expression system to produce a fusion protein and then digest it with a protecses. It's a bargain to produce a centain protein.