本文關(guān)鍵詞：Intein介導(dǎo)2-型糖尿病藥物靶點(diǎn)GPCR膜蛋白GLP-1受體的制備研究　出處：《湖北工業(yè)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 胰高血糖素-1受體 胞外域(ECD) 跨膜域(TMD) 內(nèi)含肽intein

【摘要】：胰高血糖素樣肽-1受體(glucagon like-peptide-1 receptor,GLP-1R)屬于B-簇G-蛋白偶聯(lián)受體,是設(shè)計(jì)2-型糖尿病藥物的重要靶點(diǎn)之一。GLP-1R由一個(gè)相對(duì)較大的N-端胞外域七段α-螺旋跨膜結(jié)構(gòu)域及較小的胞內(nèi)C-端域構(gòu)成。盡管通過(guò)結(jié)構(gòu)生物學(xué),蛋白質(zhì)工程等方法和手段對(duì)于GLP-1R結(jié)構(gòu)的研究取得了較大突破。但是關(guān)于其全長(zhǎng)結(jié)構(gòu)解析,配體-受體結(jié)合的分子機(jī)理和受體激活的內(nèi)在機(jī)制尚不清楚。因此,相關(guān)研究需要制備高質(zhì)量GLP-1R蛋白樣品以供蛋白結(jié)構(gòu)生物學(xué)研究。目前,常用的制備膜蛋白方法主要包括大腸表達(dá)系統(tǒng)酵母表達(dá)系統(tǒng)細(xì)胞表達(dá)系統(tǒng),桿狀病毒表達(dá)系統(tǒng)及無(wú)細(xì)胞表達(dá)系統(tǒng)。其中,后三種方法在制備膜蛋白方面成本高,表達(dá)量低等。因此,我們綜合了原核表達(dá)系統(tǒng)和真核表達(dá)系統(tǒng)的優(yōu)勢(shì)(周期短,產(chǎn)量高等),建立一種利用微生物蛋白表達(dá)系統(tǒng)制備全長(zhǎng)膜蛋白GLP-1R的方法。首先,運(yùn)用畢赤酵母表達(dá)系統(tǒng)制備GLP-1R胞外域(ECD),其優(yōu)勢(shì)在于所表達(dá)的ECD能夠在真核系統(tǒng)氧化還原條件下形成正確的蛋白構(gòu)象折疊。利用pPICZaA作為出發(fā)載體,引入多個(gè)標(biāo)簽基因(GST,intein)構(gòu)建重組載體pPICZaA-S。通過(guò)甲醇誘導(dǎo)ECD融合蛋白分泌表達(dá)。其次,通過(guò)構(gòu)建pMFH-TMD重組質(zhì)粒,利用IPTG誘導(dǎo)大腸桿菌制備GLP-1R跨膜域(TMD)。其優(yōu)勢(shì)在于選用的MFH作為融合蛋白表達(dá)TMD主要是以包涵體形式存在,避免胞內(nèi)蛋白酶的降解。所表達(dá)的融合蛋白含有MFH標(biāo)簽,TEV酶切位點(diǎn)以及半胱氨酸并利用表面活性劑來(lái)溶解和純化融合蛋白。在一定表面活性劑條件下,可通過(guò)TEV酶切割并釋放出含有游離半胱氨酸(Cys)的目標(biāo)蛋白TMD。最后,胞外域ECD和跨膜域TMD在intein介導(dǎo)下自發(fā)形成新的硫脂鍵實(shí)現(xiàn)兩部分蛋白的體外連接。本實(shí)驗(yàn)研究中,一方面,構(gòu)建了重組載體pMFH-TMD在大腸桿菌BL21pLysS中高效表達(dá)。通過(guò)優(yōu)化表達(dá)條件表明18℃,0.5 m MIPTG誘導(dǎo)20 h效果較佳;而且進(jìn)一步探索表明SDS能夠較好的溶解和純化融合蛋白MFH-TMD。在低含量SDS情況下,TEV酶仍然具有一定的切割活性。另一方面,構(gòu)建了重組載體pPICZaA-S并將該重組基因整合到畢赤酵母X33中。在0.5%甲醇誘導(dǎo)下,該融合蛋白能夠較好的分泌于發(fā)酵液中。最后,利用透析除鹽和鎳柱純化實(shí)現(xiàn)目的蛋白的富集。本研究提供了一種高效制備膜蛋白GLP-1R的方法。該方法不僅可以為其他膜蛋白制備提供了新型策略。而且能夠?yàn)樾》肿铀幬锏暮Y選提供理論依據(jù)。
[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.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R91

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本文編號(hào)：1338563