2006 Fiscal Year Final Research Report Summary
A large-scale analysis of protein-protein interactions using Escherichia coli proteome chips
| Project/Area Number |
17201042
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied genomics
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| Research Institution | Keio University |
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Principal Investigator |
DOI Nobuhide Keio University, Faculty of Science and Technology, Assistant Professor, 理工学部, 講師 (50327673)
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| Co-Investigator(Kenkyū-buntansha) |
YANAGAWA Hiroshi Keio University, Faculty of Science and Technology, Professor, 理工学部, 教授 (40327672)
MORI Hirotada Nara Institute of Science and Technology, Research and Education Center for Genetic Information, Professor, 遺伝子教育研究センター, 教授 (90182203)
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| Project Period (FY) |
2005 – 2006
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| Keywords | Proteome / Microarrays / Bacterial genome / Fluorescence labeling / Protein-protein interaction / Gene network / Bioinformatics / Escherichia coli |
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| Research Abstract |
Although protein chips are powerful tools for high-throughput analysis of protein-protein interactions (PPIs), a large-scale PPI analysis using proteome chips has not been reported yet. Here we report the construction and application of Escherichia coli proteome chips for detection of PPIs.
First we optimized the condition of PPI analysis using model E. coli proteins with regard to immobilization of proteins expressed in E. coli and detection with fluorescently labeled proteins synthesized in a cell-free system. We prepared proteome chips containing 4337 E. coli open reading frames (ORFs) and successfully detected known PPIs.
Next we attempted to perform high-throughput fluorescence labeling of various proteins in a multi-well format. We prepared template DNA of 162 E. colt genes by PCR and synthesized fluorescently labeled proteins with the PURE E. coil reconstituted system containing Cy3-dC-puromycin in 96-well microplates. One hundred and forty-five out of 162 full- length proteins (-90%) were successfully labeled with Cy3.
Finally we performed a large-scale analysis of E. colt PPIs. Overexpressed products from 4337 ORFs of E. call were printed on-100 slides in duplicate or quadruplicate, and each slide was probed by the Cy3-labeled proteins. As a result, total of 1014 PPIs including novel interactions were detected at high signal-noise ratios. When arbitrary chosen PPIs were tested by pull-down assays,-50% of PPIs were verified. These results indicate that the method established in this study should be useful to study the large-scale analysis of bacterial PPI networks.
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