A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli
A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli
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DOI：
10.1038/nmeth1148
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发表时间：
2008-01-01
期刊：
影响因子：
48
通讯作者：
Zhu, Heng
中科院分区：
文献类型：
--
作者：
Chen, Chien-Sheng;Korobkova, Ekaterina;Zhu, Heng
尽管许多基因组已被解码，但仅报道了芽殖酵母的包含单独纯化的蛋白质的蛋白质组芯片，这主要是因为高通量蛋白质纯化的复杂性和困难性。为了促进原核生物中的蛋白质组学研究，我们开发了一种高通量蛋白质纯化方案，使我们能够在10 h内纯化大肠杆菌K12菌株编码的4，256种蛋白质。然后将纯化的蛋白质点在载玻片上以产生E. coli蛋白质组芯片。我们使用这些芯片来开发用于识别参与识别DNA中潜在碱基损伤的蛋白质的检测方法。通过使用一组DNA探针，每个探针含有一个错配的碱基对或一个脱碱基位点，我们发现了少量的蛋白质，可以识别每种类型的探针具有高亲和力和特异性。我们进一步评估了这些蛋白质，YbaZ和YbcN，通过生化分析。对含有特异性修饰的DNA探针的文库的组装和E.大肠杆菌蛋白质组芯片具有揭示蛋白质和核酸之间的重要相互作用的潜力，这些相互作用是耗时的并且难以使用其他技术检测。
Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.