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SSB protein from Streptomyces coelicolor and its interactions with other proteins

Single stranded DNA binding proteins (SSBs) are essential proteins in the cells of all living organisms. These proteins are involved in DNA recombination, replication and repair. Crystal structure of SSB from the multicellular spore producing bacterium Streptomyces coelicolor has recently been solved. This structure seems to be the most stable in comparison to other structurally characterized SSBs. SSB proteins from most procaryotic species exist as tetramers which binds to ssDNA through structurally conserved folding motifs (OB folds). SSB proteins also posses conserved C-terminal domain which has role in interacting with other proteins. It has been shown that SSB protein from Escherichia coli interact with at least 14 different proteins. There is no data on interactions of the S. coelicolor SSB with other cell proteins. Therofore, the aim of this study was to identify proteins that could interact with the SSB during the growth of the S. coelicolor. As a part of our deep interest to understand tyrosine phosphorylation of SSB protein and biological significance of SSB modification we have special interest in identifying tyrosine kinase involved in this process. TAP (tandem affinity purification) tehnology has been used to purify SSB and its interacting partners from S. coelicolor. This two step purification allowes recovery of the protein complexes under native conditions. Sequences coding for two tags (protein A and Calmodulin binding peptide, CBP) separated with TEV protease cleavage site have been cloned on 5' terminus of ssb gene previously inserted into sequencing vector pGEM-T. Such construct was transformed into S. coelicolor, and protein complexes formed of SSB and unkown interacting partners were isolated from liquid culture in exponential phase of growth. The cell extract has been passed through IgG sepharose column which specifically binds to protein A. Bound recombinant protein was cleaved with TEV protease and applied to Calmodulin sepharose which binds CBP in presence of Ca2+ ions. SSB and its interacting proteins were eluted with the addition of chelating agent EGTA. Proteins were precipitated with TCA and separated with preparative SDS-PAGE. Nine visibile bands were excised and extracted from the gel slices and mass spectrometry analyses has been performed.

SSB; tandem affinity chromatography; Streptomyces ceolicolor

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