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

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.

Lactoferricin B Inhibits the Phosphorylation of the Two-Component System Response Regulators BasR and CreB

“…The E. coli Proteome Chip Assays and Bioinformatics Analysis-We employed a high-throughput E. coli protein microarray (22) to systematically identify the possible target proteins of Lfcin B. We chose DyLight™ 649 labeled streptavidin as our signal reporter for biotinylated Lfcin B and Cecropin P1.…”

“…Fabrication of the E. coli K12 Proteome Chips-For the studies of the bacterial proteome, we have constructed the E. coli K12 proteome microarray (22). In short, the E. coli K12 ASKA library (23) was first incubated with 2 ϫ Luria Broth (LB) medium containing 30 g/ml chloramphenicol in 96 DeepWell TM plates (Nunc) at 37°C overnight.…”

“…Using E. coli proteome microarrays, we discovered that Lfcin B strongly bound two TCS response regulators (21,22). We further characterized the relationships between these two proteins with Lfcin B by in vitro and in vivo analysis.…”

Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips

“…Fabrication of E. coli K12 Proteome Chip-The high throughput protein expression, protein purification, and protein printing were modified from the previous study (21). Briefly, we expressed and purified E. coli K12 proteins in 96-well plate format and subsequently printed the proteome microarray.…”

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation–π interactions

“…3, 11, and 12) are highly homologous to AGTs but have a different amino acid (typically Trp or Ala) in place of the nucleophilic active site Cys. ATL proteins retain the ability to bind single-stranded or duplex DNA containing O 6 -alkylguanine, and although unable to undertake the de-alkylative repair reaction, they protect against the adverse effects of DNA alkylation damage by downstream recruitment of nucleotide excision repair (NER) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”