蛋白激发子PebC1及其复合体的表达纯化及初步结构解析
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摘要
蛋白激发子是一类源自真菌的能够诱导植物抗性的蛋白,具有促进植物代谢和生长,增强植物抗病、抗逆能力的活性。PebC1是本实验室从灰霉菌(Botrvtis cinerea)中分离得到的蛋白激发子(Zhang, et al., 2010),为了深入阐明PebC1的功能机理,本文对PebC1蛋白和能与其相互作用的灰霉菌βNAC蛋白分别用原核和真核表达系统进行了表达,并进一步进行了PebC1与bcβNAC复合体蛋白表达;在此基础上对表达蛋白进行纯化,对PebC1蛋白进行了初步结构解析。主要结果如下:
1、通过对PebC1蛋白序列进行信息学分析,推测其是NAC(Nascent polypeptide Associated Complex)蛋白家族中αNAC同源蛋白。通过查询基因组数据库,得到灰霉菌中βNAC(bcβNAC)的编码基因egd1的基因序列,为深入研究PebC1蛋白的结构打下了基础。
2、构建了PebC1及bcβNAC蛋白原核和真核表达系统,蛋白表达表明PebC1及bcβNAC蛋白适合用原核系统表达。将pebC1基因和egd1基因分别克隆到原核表达载体pET-30 His和pET-30 His+MBP上分别进行原核表达;同时将pebC1基因和egd1基因分别克隆到pFastBac His+GST载体上,得到重组的杆状病毒并分别进行真核表达。
3、成功表达了PebC1和bcβNAC复合体蛋白。在原核表达系统中,将pebC1基因和egd1基因分别克隆到pETDuet-1双表达质粒的两个多克隆位点上,进行复核体蛋白表达,并得到超表达的复合体蛋白;在真核表达系统中,利用带有pebC1基因和egd1基因的两种重组病毒同时感染Hi5细胞进行真核表达,但由于PebC1被降解,不能表达复合体蛋白。
4、完成对PebC1、bcβNAC以及复合体蛋白纯化条件的摸索。利用亲和层析、离子交换层析和分子筛层析的三步法进行纯化,摸索纯化条件,并得到高纯度的目的蛋白,为后续实验奠定基础。
5、通过小角散射实验得到PebC1和bcβNAC的点阵结构模型。利用纯化的PebC1和bcβNAC蛋白进行小角散射实验,收集PebC1和带MBP标签的bcβNAC在小角区域的散射信号,对实验数据进行处理后得到蛋白的点阵模型。结合对PebC1的NAC和UBA结构域(Ubiquitin Associated domain)的同源建模结构的分析,表明PebC1呈S型对称分布,NAC二聚体形成凹陷的β桶状结构,UBA结构域悬挂在侧边。mbp-bcβNAC的点阵模型大体可分成两部分,Part 2呈直径50?的圆柱状;Part 1为厚度30?扁平状,同MBP蛋白的空间结构一致,由此推测MBP位于Part 1。bcβNAC的精细结构还需进一步实验测定。
6、筛选得到复合体蛋白的初始结晶条件。采用气相扩散坐滴法,在20℃条件下筛选复合体蛋白的结晶条件,并在Hampton Research公司的Crystal Screen II的19#和MolecularDimensions MD1-02 34#条件下得到孪晶,为最终获得高质量单晶,用X射线衍射分析得到三维结构奠定了重要基础。
Protein elicitor is a type of protein derived from pathogenic fungi, which can interact with plant and induce plant defense response, accelerate plant metabolism and promote plant growth. PebC1 is a novel protein elicitor derived from Botrvtis cinerea by our laboratory. In this study, we did cloning and expression of PebC1 and bcβNAC which have an interaction with PebC1 with prokaryotic and eukaryotic expression system. Then the coexpression of the two proteins was made. After purification, primary structure analysis of PebC1 and bcβNAC protein was done by a serials assay. The main results are as follows.
The amino acid sequence of PebC1 was analysed by bioinformatic tools and cloned the gene egd1 witch encoded the bcβNAC based on the online database.
The prokaryotic expression system is suitable for PebC1 and bcβNAC protein expression. Amplified gene pebC1 and egd1from Botrvtis cinerea were ligated to expression vector pET-30His and pET-30His+MBP respectively. The recombinant plasmids were transformed into expression strain Rosetta. The two genes were also inserted into pFastBac donor vectors, and prepared recombinant baculovirus stocks respectively. Then the recombinant baculovirus were used to transfect High Five(Hi5)cells for expression.
The prokaryotic expression system is suitable for PebC1 and bcβNAC complex protein expression. The gene egd1and pebC1 were inserted into the mult-cloning site 1 and mult-cloning site 2 of pETDuet-1 vector respectively. Then the recombinant plasmid was transformed into expression strain Rosetta for coexpression. The two recombinant baculovirus also used to transfected Hi5 cells simultaneously for coexpression. But no complex protein can get due to the rapid digestion of PebC1 in insect cells. The purifications of all those proteins were realized. Those proteins were purified by affinity chromatography. Then HiTrap Q HP column was used for anion exchange chromatography. Finally size exclusion chromatography was performed through Superdex 200 column. The highly purified proteins were prepared for next experiments.
The dot matrix structure of PebC1 and mbp-bcβNAC was determined by small angle scattering analysis. According to the homology model of NAC domain and UBA domain, we assigned the homodimer of NAC domain formed into aβbarrel in the central of the protein. Other parts except the UBA domain tightly surround the barrel. The UBA domains were hanged by the side. According to the dot matrix model of mbp-bcβNAC, the structure was divided into two parts: Part 2 forms into a cylindrical with 50A diameter; Part 1 forms into platykurtic with 30? depth which match the structure of MBP protein. So MBP protein was assigned into Part 1. But the exactly structure of bcβNAC remain unclear.
Twinned crystals of complex protein were obtained. Purified heterodimer protein of PebC1 and bcβNAC was set up to grow crystals by sitting-drop vapor-diffusion method at 20℃. Twinned crystals were emerged in two conditions, which were Crystal Screen II 19# and MolecularDimensions MD1-02 34#. The results facilitate the structure research of PebC1 and its complex protein with bcβNAC.
1、通过对PebC1蛋白序列进行信息学分析,推测其是NAC(Nascent polypeptide Associated Complex)蛋白家族中αNAC同源蛋白。通过查询基因组数据库,得到灰霉菌中βNAC(bcβNAC)的编码基因egd1的基因序列,为深入研究PebC1蛋白的结构打下了基础。
2、构建了PebC1及bcβNAC蛋白原核和真核表达系统,蛋白表达表明PebC1及bcβNAC蛋白适合用原核系统表达。将pebC1基因和egd1基因分别克隆到原核表达载体pET-30 His和pET-30 His+MBP上分别进行原核表达;同时将pebC1基因和egd1基因分别克隆到pFastBac His+GST载体上,得到重组的杆状病毒并分别进行真核表达。
3、成功表达了PebC1和bcβNAC复合体蛋白。在原核表达系统中,将pebC1基因和egd1基因分别克隆到pETDuet-1双表达质粒的两个多克隆位点上,进行复核体蛋白表达,并得到超表达的复合体蛋白;在真核表达系统中,利用带有pebC1基因和egd1基因的两种重组病毒同时感染Hi5细胞进行真核表达,但由于PebC1被降解,不能表达复合体蛋白。
4、完成对PebC1、bcβNAC以及复合体蛋白纯化条件的摸索。利用亲和层析、离子交换层析和分子筛层析的三步法进行纯化,摸索纯化条件,并得到高纯度的目的蛋白,为后续实验奠定基础。
5、通过小角散射实验得到PebC1和bcβNAC的点阵结构模型。利用纯化的PebC1和bcβNAC蛋白进行小角散射实验,收集PebC1和带MBP标签的bcβNAC在小角区域的散射信号,对实验数据进行处理后得到蛋白的点阵模型。结合对PebC1的NAC和UBA结构域(Ubiquitin Associated domain)的同源建模结构的分析,表明PebC1呈S型对称分布,NAC二聚体形成凹陷的β桶状结构,UBA结构域悬挂在侧边。mbp-bcβNAC的点阵模型大体可分成两部分,Part 2呈直径50?的圆柱状;Part 1为厚度30?扁平状,同MBP蛋白的空间结构一致,由此推测MBP位于Part 1。bcβNAC的精细结构还需进一步实验测定。
6、筛选得到复合体蛋白的初始结晶条件。采用气相扩散坐滴法,在20℃条件下筛选复合体蛋白的结晶条件,并在Hampton Research公司的Crystal Screen II的19#和MolecularDimensions MD1-02 34#条件下得到孪晶,为最终获得高质量单晶,用X射线衍射分析得到三维结构奠定了重要基础。
Protein elicitor is a type of protein derived from pathogenic fungi, which can interact with plant and induce plant defense response, accelerate plant metabolism and promote plant growth. PebC1 is a novel protein elicitor derived from Botrvtis cinerea by our laboratory. In this study, we did cloning and expression of PebC1 and bcβNAC which have an interaction with PebC1 with prokaryotic and eukaryotic expression system. Then the coexpression of the two proteins was made. After purification, primary structure analysis of PebC1 and bcβNAC protein was done by a serials assay. The main results are as follows.
The amino acid sequence of PebC1 was analysed by bioinformatic tools and cloned the gene egd1 witch encoded the bcβNAC based on the online database.
The prokaryotic expression system is suitable for PebC1 and bcβNAC protein expression. Amplified gene pebC1 and egd1from Botrvtis cinerea were ligated to expression vector pET-30His and pET-30His+MBP respectively. The recombinant plasmids were transformed into expression strain Rosetta. The two genes were also inserted into pFastBac donor vectors, and prepared recombinant baculovirus stocks respectively. Then the recombinant baculovirus were used to transfect High Five(Hi5)cells for expression.
The prokaryotic expression system is suitable for PebC1 and bcβNAC complex protein expression. The gene egd1and pebC1 were inserted into the mult-cloning site 1 and mult-cloning site 2 of pETDuet-1 vector respectively. Then the recombinant plasmid was transformed into expression strain Rosetta for coexpression. The two recombinant baculovirus also used to transfected Hi5 cells simultaneously for coexpression. But no complex protein can get due to the rapid digestion of PebC1 in insect cells. The purifications of all those proteins were realized. Those proteins were purified by affinity chromatography. Then HiTrap Q HP column was used for anion exchange chromatography. Finally size exclusion chromatography was performed through Superdex 200 column. The highly purified proteins were prepared for next experiments.
The dot matrix structure of PebC1 and mbp-bcβNAC was determined by small angle scattering analysis. According to the homology model of NAC domain and UBA domain, we assigned the homodimer of NAC domain formed into aβbarrel in the central of the protein. Other parts except the UBA domain tightly surround the barrel. The UBA domains were hanged by the side. According to the dot matrix model of mbp-bcβNAC, the structure was divided into two parts: Part 2 forms into a cylindrical with 50A diameter; Part 1 forms into platykurtic with 30? depth which match the structure of MBP protein. So MBP protein was assigned into Part 1. But the exactly structure of bcβNAC remain unclear.
Twinned crystals of complex protein were obtained. Purified heterodimer protein of PebC1 and bcβNAC was set up to grow crystals by sitting-drop vapor-diffusion method at 20℃. Twinned crystals were emerged in two conditions, which were Crystal Screen II 19# and MolecularDimensions MD1-02 34#. The results facilitate the structure research of PebC1 and its complex protein with bcβNAC.
引文
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2.卢光莹,华子千.生物大分子晶体学基础.北京:北京大学出版社, 1995: 2-7.
3.邱德文.微生物蛋白农药研究进展.中国生物防治, 2004, 20(2):91-94.
4.王大成.后基因组时代中的结构生物学.生物化学与生物物理进展, 1998, 27(4): 340-344.
5.阎隆飞,孙之荣.蛋白质分子结构.北京:清华大学出版社.1999.
6.张浩,毛秉智,李晓霞,王全立,冯起,刘晓达.基因重组人血红蛋白质的纯化.中国生化药物杂质, 2000, 21(6):274-277.
7.章元寿.植物病理生理学.江苏:江苏科学技术出版社, 1996:245-263.
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