Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of<i>Escherichia coli</i>; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure

Abstract

The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography.Both receptor proteins ran as a single protein band on SDS-PAGE.Their apparent M r values were 74000 and 76 000, respectively.The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloaein DF13 were determined.Values of 2.0 10 s M -1 and 1.0 x 109 M -1, respectively, were found.The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced.The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues.The mature protein has an M r of 77 345.