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mLST8在大肠杆菌中的表达、纯化及鉴定
引用本文:朱传智,郝娟,黄香玉,赵雅贞,何秀云,石炳毅.mLST8在大肠杆菌中的表达、纯化及鉴定[J].生物技术通讯,2012,23(4):493-496.
作者姓名:朱传智  郝娟  黄香玉  赵雅贞  何秀云  石炳毅
作者单位:1. 西南大学生命科学学院,重庆,400715
2. 解放军第三○九医院器官移植与免疫调节北京市重点实验室,北京,100091
3. 西南大学生命科学学院,重庆 400715;解放军第三○九医院器官移植与免疫调节北京市重点实验室,北京 100091
摘    要:目的:表达并纯化mLST8蛋白。方法:PCR扩增mLST8的编码cDNA,克隆到pET-28a(+)表达载体,将重组质粒pET-28a-mLST8转化大肠杆菌BL21(DE3)感受态细胞,在IPTG诱导下表达目的蛋白;提取包涵体,用Ni2+亲和层析纯化目的蛋白,稀释和透析相结合进行复性,对复性蛋白进行阴离子交换层析、分子筛层析,将纯的复性mLST8进行肽指纹质谱鉴定和圆二色谱分析。结果:酶切和DNA测序证明pET-28a-mLST8表达质粒构建无误,并在大肠杆菌中得到高效表达;通过Ni2+亲和层析、复性、离子交换层析和分子筛层析获得了较高纯度的复性蛋白,肽指纹质谱鉴定为mLST8;mLST8蛋白的二级结构α螺旋为18.2%,β折叠为52.3%(其中平行结构为12.1%,反向平行结构为40.2%),β转角为20.7%,无规则卷曲为39.9%]表明其为典型的β折叠结构。结论:在大肠杆菌中表达了重组mLST8蛋白,复性获得了二级结构准确的mLST8,为进一步研究mLST8的晶体结构与功能奠定了基础。

关 键 词:mLST8  原核表达  纯化  肽指纹质谱  圆二色谱

Expression,Purification and Identification of Recombinant mLST8 in Escherichia coli
ZHU Chuan-Zhi , HAO Juan , HUANG Xiang-Yu , ZHAO Ya-Zhen , HE Xiu-Yun , SHI Bing-Yi.Expression,Purification and Identification of Recombinant mLST8 in Escherichia coli[J].Letters in Biotechnology,2012,23(4):493-496.
Authors:ZHU Chuan-Zhi  HAO Juan  HUANG Xiang-Yu  ZHAO Ya-Zhen  HE Xiu-Yun  SHI Bing-Yi
Institution:1.School of Life Sciences,Southwest University,Chongqing 400715;2.Beijing Key Laboratory of Transplantation and Immune Regulation,309th Hospital of PLA,Beijing 100091;China
Abstract:Objective: To clone,express,purify and acquire high purification and correct refolding of mLST8.Methods: The gene encoded mLST8 was amplified with PCR and cloned into pET-28a(+) expression vector.The recombinant plasmid pET-28a-mLST8 was transformed into E.coli BL21(DE3) competence.The recombinant E.coli BL21(DE3) was induced by IPTG.Inclusion bodies were extracted,dissolved in 8 mol/L urea and purified by Ni2 + affinity chromatography.The renatured mLST8 with dilution and dialysis method was purified by anion-ex change chromatography and gel filtration chromatography.The correctly folded mLST8 was identified with peptide mass fingerprinting and circular dichroism spectra analysis.Results: Restrict endonuclease analysis and DNA se quence were testified that mLST8 gene was successfully inserted into pET-28a(+).Recombinant mLST8 protein was highly expressed as inclusion bodies in E.coli.The secondary structure of mLST8 was composed of 18.2% of α-helix,52.3% of β-sheet(12.1% of the parallel structure,40.2% of the antiparallel structure),20.7% of β-turn and 39.9% of random coil.Conclusion: mLST8 was successfully expressed in E.coli,and refolded mLST8 with cor rect secondary structure was help to further study its crystal structure and function.
Keywords:mLST8  prokaryotic expression  purification  peptide mass fingerprinting  circular dichroism
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