本文關(guān)鍵詞：果蠅烯醇酶的結(jié)構(gòu)生物學(xué)研究　出處：《青島科技大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 果蠅烯醇酶 純化 結(jié)晶 結(jié)構(gòu)

【摘要】：烯醇酶在細(xì)胞的許多生理生化功能方面都發(fā)揮著重要的調(diào)節(jié)作用,烯醇酶的差異化表達(dá)還被認(rèn)為是一些病理狀態(tài)的標(biāo)志,諸如癌癥、阿爾茲海默癥、類(lèi)風(fēng)濕性關(guān)節(jié)炎等。本文以果蠅總cDNA為模板,克隆了烯醇酶基因,序列分析表明獲得的基因與GenBank報(bào)道的果蠅烯醇酶序列完全一致。將含有該基因的表達(dá)載體轉(zhuǎn)化大腸桿菌BL21感受態(tài)后,經(jīng)SDS-PAGE檢測(cè)證實(shí),目的基因在BL21中得到了表達(dá),獲得了大小為47KD的蛋白,并具有較高的表達(dá)量,符合預(yù)期結(jié)果。在親和層析純化的基礎(chǔ)上,進(jìn)一步經(jīng)過(guò)脫鹽處理和凝膠過(guò)濾層析后獲得純度非常高的電泳純目的蛋白,符合下一步的結(jié)晶要求。用座滴氣相擴(kuò)散法對(duì)果蠅烯醇酶蛋白進(jìn)行大規(guī)模晶體篩選,初篩條件為10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4。晶體形成后再進(jìn)一步優(yōu)化,最終的優(yōu)化條件為10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4,0.1 M CdCl2。優(yōu)化后得到顆粒較大,生長(zhǎng)良好的單晶。使用X射線晶體衍射的方法對(duì)優(yōu)化后的晶體進(jìn)行初步解析,在上海同步輻射光源的BLl7U光束工作站收集晶體衍射數(shù)據(jù),用HKL2000進(jìn)行數(shù)據(jù)處理。其分辨率可達(dá)2.1?,屬于C121空間群,晶胞參數(shù)a=168.746?,b=119.210,c=103.191?,α=γ=90.00°,?=114.28°。果蠅烯醇酶的蛋白晶體結(jié)構(gòu)由分子置換的方法確定。其結(jié)構(gòu)解析表明,和其他大多數(shù)烯醇酶一樣,果蠅烯醇酶能夠在一個(gè)二聚體界面形成同型二聚體。果蠅烯醇酶的蝶形二聚體具有保守殘基,并且通過(guò)離子鍵、氫鍵和疏水相互作用維持。它有一個(gè)保守的催化區(qū)域及一個(gè)帶有不穩(wěn)定L1環(huán)的相對(duì)活躍的構(gòu)象。其活性位點(diǎn)被一個(gè)鎘離子,兩個(gè)鈷離子和一個(gè)硫酸根離子占據(jù),硫酸鹽取代烯醇酶底物磷酸基團(tuán)并且和殘基Ala106,Arg440以及Ser441形成鹽橋和氫鍵。與其他物種烯醇酶不同的是,果蠅烯醇酶在N末端有一超長(zhǎng)區(qū)域,這一超長(zhǎng)區(qū)域可能對(duì)其功能的發(fā)揮起著調(diào)節(jié)作用。本文首次對(duì)果蠅的烯醇酶(69-500)進(jìn)行了結(jié)構(gòu)解析,為進(jìn)一步研究烯醇酶的結(jié)構(gòu)和功能方面奠定了基礎(chǔ)。
[Abstract]:Enolase plays an important role in regulating many physiological and biochemical functions of cells. The differential expression of enolase is also considered to be a marker of pathological state, such as cancer, Alzheimer's disease. In this paper, the enolase gene was cloned from the total cDNA of Drosophila melanogaster. Sequence analysis showed that the obtained gene was completely consistent with the sequence of Drosophila melanogaster enolase reported by GenBank. The expression vector containing the gene was transformed into E. coli BL21 competent state. It was confirmed by SDS-PAGE that the target gene was expressed in BL21, and a protein of 47KD was obtained. In accordance with the expected results, on the basis of affinity chromatography purification, after further desalination and gel filtration chromatography, a very high purity electrophoresis pure protein was obtained. According to the requirements of the next step of crystallization, the large scale crystal screening of drosophila enolase protein was carried out by gas phase diffusion method. The initial screening condition was 10 mm CoCl2 + 0.1 M MES pH6.5. The crystal was further optimized after the crystal formation, and the final optimization condition was 10 mm CoCl2 + 0.1 M MES pH6.5. 1.9 NH _ 4H _ 4SO _ 4SO _ 4O _ (1) M CdCl _ (2). The particle size was larger after optimization. The X-ray diffraction method was used to analyze the optimized crystal and the diffraction data were collected at the BLl7U beam workstation of Shanghai Synchrotron radiation source. Data processing with HKL2000. Its resolution can reach 2.1? , belonging to C121 space group, unit cell parameter a168.746? BX 119.210CU 103.191? , 偽 = 90.00 擄? The protein crystal structure of Drosophila enolase was determined by the method of molecular replacement. The structural analysis showed that the protein crystal structure was the same as most other enolases. Drosophila enolase can form a homodimer at the interface of a dimer. The butterfly dimer of Drosophila enolase has conserved residues and passes through ionic bonds. The hydrogen bond and hydrophobic interaction are maintained. It has a conserved catalytic region and a relatively active conformation with unstable L1 ring. Its active site is a cadmium ion. Two cobalt ions and one sulfate ion occupy the sulfate-substituted enolase substrate phosphate group and the residue of Ala106. Arg440 and Ser441 form salt bridges and hydrogen bonds. Unlike other species enolase, Drosophila enolase has a very long region at the N end. This super-long region may play a role in regulating its function. In this paper, the structure of the enolase of Drosophila melanogaster 69-500 was analyzed for the first time. It lays a foundation for further study on the structure and function of enolase.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q55

【參考文獻(xiàn)】

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1 王瑩;張t，

本文編號(hào)：1422811