| Glucagon-like peptide-1 receptor(GLP-1R)belongs to class B family of G-protein-coupled receptor,which is one of the important targets for designing type 2diabetes mellitus.GLP-1R consists of a relatively large N-terminal extracellular domain,a seven transmembrane helix domain,and a smaller intracellular C-terminal domain.The breakthrough on GLP-1R structure and functions have been made via structural biology and protein engineering.However,it is still unknow on the analysis of its full-length structure,the molecular mechanism of receptor activation.However,the intrinsic mechanism of molecular mechanism and receptor activation of ligand-receptor binding is unclear about its full-length structure analysis.Therefore,the preparation of high quality GLP-1R protein samples were obtained by related studies for structure biology research.At present,the commonly used method for preparing membrane proteins mainly includes E.coli expression system,yeast expression system,cell expression system,bacovirus expression system and cell-free expression system.The latter three methods for preparation of membrane protein are the high cost,low-yield expression and so on.Therefore,we summrise the advantages of prokaryotic expression system and eukaryotic expression system(short generation and high-yield),and establish a method for the preparation of membrane protein full-length GLP-1R via a microbial expression system.Firstly,the GLP-1R extracellular domain(ECD)was obtained using the yeast expression system.The advantage is that the expressed ECD can form the correct protein conformation fold under the conditions of eukaryotic system redox.The recombinant vector pPICZaA-S was constructed by using pPICZa A as the starting vector and introducing multiple tag genes(GST,intein).The expression of secreted protein was produced by methanol induced.Secondly,the GLP-1R transmembrane domain(TMD)was prepared using the E.coli expression system.The advantage using MFH as a fusion protein expression TMD is in the form of inclusion body and avoid intracellular protease degradation.The expressed fusion protein contains the MFH tag,the TEV cleavage site,and the cysteine and dissolve and purify the fusion protein.Under certain surfactant conditions by detergent,the target protein TMD containing free cysteine(Cys)can be cleaved and released by TEV protease in the low-level detergent.Finally,the extracellular domain ECD and transmembrane domain TMD spontaneously form a new thiozole bond by intein mediated to achieve the ligation of the two-part protein.In this study,on one hand,the recombinant vector pMFH-TMD was constructed and expressed efficiently in E.coli BL21 pLysS.The optimization of the expression conditions show that the expression level is best at 18? for 20 h in the presence of 0.5m MIPTG and the fusion protein is in the form of inclusion bodies;The further study show that SDS can better dissolve and purify the fusion protein MFH-TMD.In the low levels of SDS,TEV protease still have a certain cleavage activity,but the activity isweak.On the other hand,the recombinant vector pPICZaA-S was constructed and the recombinant gene was integrated into P.pastoris X33.Under the induction of 0.5%methanol,the fusion protein can be secreted better in the fermentation broth.Finally,the enrichment of the target protein was achieved by dialysis desalting and nickel column purification.This study provides a new method for the efficient preparation of membrane protein GLP-1R.This method not only provides a new strategy for the preparation of other membrane proteins,but also provide a theoretical basis for the screening of small molecule drugs... |
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