Salvaging Pyrococcus furiosus protein targets at SECSG.
Salvaging Pyrococcus furiosus protein targets at SECSG.
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拯救狂怒火球菌蛋白质目标在 SECSG。
DOI：
10.1007/s10969-005-5242-x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Newton,MGa
中科院分区：
文献类型：
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作者：
Liu,Zhi-Jie;Shah,AshitK;Habel,JeffE;Ng,JosephD;Kataeva,Irina;Xu,Hao;Horanyi,Peter;Yang,Hua;Chang,Jessie;Zhao,Min;Huang,Lei;Chang,Sue;Tempel,Wolfram;Chen,Lirong;Zhou,Weihong;Lee,Doowon;Lin,Dawei;Zhang,Hua;Newton,MGa
来自激烈火球菌编码区的蛋白质是东南结构基因组学合作实验室（SECSG）进行三维X射线和NMR结构测定的目标。在该生物体的2200个开放阅读框（ORF）中，克隆了220个蛋白质靶点，并在高通量（HT）重组系统中表达，用于晶体学研究。然而，只有96个表达的蛋白质可以结晶，其中只有15个导致了结构。为了解决这个问题，SECSG最近开发了一种蛋白质生产和结晶的双层方法。在这种方法中，第一层的努力集中在生产蛋白质的newPfu（斜体？）使用高通量方法的目标。2级蛋白质生产工作通过以下方式支持1级活动：（1）生产用于进一步结晶试验的额外蛋白质，（2）生产修饰蛋白质（进一步纯化、甲基化、标签去除、硒标记等）和（3）作为失败的第1层蛋白的补救途径。从一组50Pfu蛋白中确定了9种结构，这些蛋白不能产生适合于X射线衍射分析的晶体。这些结果验证了这种方法，并建议它有其他HT晶体结构测定应用程序的应用。
Proteins derived from the coding regions ofPyrococcus furiosusare targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for newPfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins.In a recent study using this two-tiered approach, nine structures were determined from a set of 50Pfuproteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.