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In vitro DNA binding of purified CcpA protein from Lactococcus lactis IL1403

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During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.
Physical description
  • Department of Microbial Biochemistry PAS, Warszawa, Poland
  • Department of Microbial Biochemistry PAS, Warszawa, Poland
  • Department of Bioinformatics, Institute of Biochemistry and Biophysics PAS, Warszawa, Poland
  • Department of Microbial Biochemistry PAS, Warszawa, Poland
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