Spindlin1的蛋白结构解析及相互作用蛋白的寻找
发布时间：2019-05-20 09:04
【摘要】：spindlin1是ssty/spin家族成员,编码一个237个残基的多肽链。它的功能和结构基础目前所知甚少。德国学者对已经报告的spin/ssty家族成员进行了生物信息学分析,发现了一个Spin/Ssty repeat,该实验组认为这个Spin/Ssty repeat是一个新的motif、是独立的功能单位,对该motif进一步分析,预测会形成一种未经报道过的结构---four-stranded beta-structure。 我们原核表达了GST融合的Spindlin1,亲和纯化、切去GST后经阴离子交换色谱纯化,得到了高纯度的重组Spindlin1。我们采用气相悬滴法获得了可供X线衍射的晶体,经过结晶条件优化后,进行了同步辐射收集数据。衍射数据经过软件处理后我们得到了Spindlin1的空间结构,该结构数据已经提交到PDB数据库,登录号为1YDJ,目前数据还没有释放。 我们实验组用真核表达载体pEGFP-N1-spindlin1转染NIH 3T3细胞,对筛选出的稳定转染细胞进行了细胞生物学检测。因此我们在生物信息学和晶体结构的基础上设计了突变体。真核突变载体已经转染NIH 3T3细胞,正在扩大培养。原核突变载体已经交给清华大学结构生物学实验室。这部分工作将最终建立起结构一功能的关系。
[Abstract]:Spindlin1 is a member of the ssty/spin family and encodes a polypeptide chain of 237residues. Little is known about its functional and structural basis. German scholars carried out bioinformatics analysis of reported members of the spin/ssty family and found a Spin/Ssty repeat,. The experimental group considered the Spin/Ssty repeat to be a new motif, as an independent functional unit, and further analyzed the motif. Predict that there will be an unreported structure-four-stranded beta-structure. We expressed GST fusion Spindlin1, affinity purification in prokaryotic. After GST was cut off, it was purified by anion exchange chromatography, and the recombinant Spindlin1. with high purity was obtained. The crystals which can be diffracted by X-ray diffraction were obtained by gas phase suspension drop method. After the crystallization conditions were optimized, the synchrotron radiation data were collected. After the diffraction data is processed by software, we get the spatial structure of Spindlin1, and the structure data has been submitted to PDB database, the login number is 1YDJ, and the data has not yet been released. In our experimental group, NIH 3T3 cells were transfected with eukaryotic expression vector pEGFP-N1-spindlin1, and the selected stable transfected cells were detected by cell biology. Therefore, we designed mutants on the basis of bioinformatics and crystal structure. Eukaryotic mutant vector has been transformed into NIH 3T3 cells and is being cultured. The prokaryotic mutation vector has been handed over to the Laboratory of structural Biology of Tsinghua University. This part of the work will eventually establish the relationship between structure and function.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R341
本文编号：2481525
[Abstract]:Spindlin1 is a member of the ssty/spin family and encodes a polypeptide chain of 237residues. Little is known about its functional and structural basis. German scholars carried out bioinformatics analysis of reported members of the spin/ssty family and found a Spin/Ssty repeat,. The experimental group considered the Spin/Ssty repeat to be a new motif, as an independent functional unit, and further analyzed the motif. Predict that there will be an unreported structure-four-stranded beta-structure. We expressed GST fusion Spindlin1, affinity purification in prokaryotic. After GST was cut off, it was purified by anion exchange chromatography, and the recombinant Spindlin1. with high purity was obtained. The crystals which can be diffracted by X-ray diffraction were obtained by gas phase suspension drop method. After the crystallization conditions were optimized, the synchrotron radiation data were collected. After the diffraction data is processed by software, we get the spatial structure of Spindlin1, and the structure data has been submitted to PDB database, the login number is 1YDJ, and the data has not yet been released. In our experimental group, NIH 3T3 cells were transfected with eukaryotic expression vector pEGFP-N1-spindlin1, and the selected stable transfected cells were detected by cell biology. Therefore, we designed mutants on the basis of bioinformatics and crystal structure. Eukaryotic mutant vector has been transformed into NIH 3T3 cells and is being cultured. The prokaryotic mutation vector has been handed over to the Laboratory of structural Biology of Tsinghua University. This part of the work will eventually establish the relationship between structure and function.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R341
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