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Nat Methods. 2008 Jan;5(1):69-74. Epub 2007 Dec 16.

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.

Author information

1
Department of Pharmacology and Molecular Sciences & High Throughput Biology Center, Johns Hopkins University School of Medicine, Broadway Research Building 333, 733 Broadway, Baltimore, Maryland 21205, USA.

Abstract

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.

PMID:
18084297
PMCID:
PMC2576280
DOI:
10.1038/nmeth1148
[Indexed for MEDLINE]
Free PMC Article
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