Intein介导2-型糖尿病药物靶点GPCR膜蛋白GLP-1受体的制备研究

发布时间：2017-12-26 19:31

本文关键词：Intein介导2-型糖尿病药物靶点GPCR膜蛋白GLP-1受体的制备研究　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：胰高血糖素样肽-1受体(glucagon like-peptide-1 receptor,GLP-1R)属于B-簇G-蛋白偶联受体,是设计2-型糖尿病药物的重要靶点之一。GLP-1R由一个相对较大的N-端胞外域七段α-螺旋跨膜结构域及较小的胞内C-端域构成。尽管通过结构生物学,蛋白质工程等方法和手段对于GLP-1R结构的研究取得了较大突破。但是关于其全长结构解析,配体-受体结合的分子机理和受体激活的内在机制尚不清楚。因此,相关研究需要制备高质量GLP-1R蛋白样品以供蛋白结构生物学研究。目前,常用的制备膜蛋白方法主要包括大肠表达系统酵母表达系统细胞表达系统,杆状病毒表达系统及无细胞表达系统。其中,后三种方法在制备膜蛋白方面成本高,表达量低等。因此,我们综合了原核表达系统和真核表达系统的优势(周期短,产量高等),建立一种利用微生物蛋白表达系统制备全长膜蛋白GLP-1R的方法。首先,运用毕赤酵母表达系统制备GLP-1R胞外域(ECD),其优势在于所表达的ECD能够在真核系统氧化还原条件下形成正确的蛋白构象折叠。利用pPICZaA作为出发载体,引入多个标签基因(GST,intein)构建重组载体pPICZaA-S。通过甲醇诱导ECD融合蛋白分泌表达。其次,通过构建pMFH-TMD重组质粒,利用IPTG诱导大肠杆菌制备GLP-1R跨膜域(TMD)。其优势在于选用的MFH作为融合蛋白表达TMD主要是以包涵体形式存在,避免胞内蛋白酶的降解。所表达的融合蛋白含有MFH标签,TEV酶切位点以及半胱氨酸并利用表面活性剂来溶解和纯化融合蛋白。在一定表面活性剂条件下,可通过TEV酶切割并释放出含有游离半胱氨酸(Cys)的目标蛋白TMD。最后,胞外域ECD和跨膜域TMD在intein介导下自发形成新的硫脂键实现两部分蛋白的体外连接。本实验研究中,一方面,构建了重组载体pMFH-TMD在大肠杆菌BL21pLysS中高效表达。通过优化表达条件表明18℃,0.5 m MIPTG诱导20 h效果较佳;而且进一步探索表明SDS能够较好的溶解和纯化融合蛋白MFH-TMD。在低含量SDS情况下,TEV酶仍然具有一定的切割活性。另一方面,构建了重组载体pPICZaA-S并将该重组基因整合到毕赤酵母X33中。在0.5%甲醇诱导下,该融合蛋白能够较好的分泌于发酵液中。最后,利用透析除盐和镍柱纯化实现目的蛋白的富集。本研究提供了一种高效制备膜蛋白GLP-1R的方法。该方法不仅可以为其他膜蛋白制备提供了新型策略。而且能够为小分子药物的筛选提供理论依据。
[Abstract]:Glucagon like peptide -1 receptor (glucagon like-peptide-1 receptor, GLP-1R) belongs to the B- cluster G- protein coupled receptor, and it is one of the important targets for designing 2- diabetes drugs. GLP-1R is composed of a relatively large N- endpoint, seven segment alpha helix transmembrane domain and small intracellular C- end domain. Although the structure biology, protein engineering and other methods and means have made great breakthroughs in the research of GLP-1R structure. However, the molecular mechanism of ligand receptor binding and the internal mechanism of receptor activation are still unclear about its full length structure analysis. Therefore, the relevant research needs to prepare high quality GLP-1R protein samples for protein structural biology research. At present, the commonly used methods of preparing membrane proteins include the expression system of large intestine, yeast expression system, cell expression system, baculovirus expression system and cell-free expression system. Among them, the latter three methods have high cost and low expression in the preparation of membrane protein. Therefore, we synthesized the advantages of prokaryotic expression system and eukaryotic expression system (short cycle and high yield), and established a method to prepare full-length membrane protein GLP-1R by using microbial protein expression system. First, the GLP-1R extracellular domain (ECD) was prepared by Pichia pastoris expression system. The advantage is that the expressed ECD can form the correct protein conformation folding under the redox conditions of eukaryotic system. PPICZaA was used as the starting carrier and multiple label genes (GST, intein) were introduced to construct the recombinant vector pPICZaA-S. The expression of ECD fusion protein was induced by methanol. Secondly, through the construction of pMFH-TMD recombinant plasmid, the GLP-1R transmembrane domain (TMD) was prepared by using IPTG to induce Escherichia coli. The advantage of the MFH is that the expression of TMD, as a fusion protein, is mainly in the form of inclusion bodies and avoids the degradation of intracellular protease. The expressed fusion protein contains the MFH label, the TEV enzyme cutting site and cysteine and uses the surfactant to dissolve and purify the fusion protein. Under certain surfactants, the target protein TMD containing free cysteine (Cys) can be cut through the TEV enzyme. Finally, the extracellular domain ECD and transmembrane domain TMD spontaneously form new sulfur fat bonds mediated by intein to realize the in vitro connection of two parts of the protein. In this experimental study, on the one hand, a recombinant vector pMFH-TMD was constructed to express efficiently in Escherichia coli BL21pLysS. The optimized expression conditions showed that the effect of 20 h was better at 18, 0.5 m MIPTG, and further exploration showed that SDS could better dissolve and purify the fusion protein MFH-TMD. Under the condition of low content of SDS, the TEV enzyme still has a certain cutting activity. On the other hand, the recombinant vector pPICZaA-S was constructed and the recombinant gene was integrated into Pichia pastoris X33. Under the induction of 0.5% methanol, the fusion protein was well secreted in the fermentation broth. Finally, the enrichment of the target protein was achieved by the purification of dialysate and the purification of the nickel column. This study provides a highly efficient method for the preparation of membrane protein GLP-1R. This method can not only provide a new strategy for the preparation of other membrane proteins. And it can provide a theoretical basis for the screening of small molecule drugs.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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