Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure |
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| Authors: | Willy JA Krone Freek Stegehuis Gregory Koningstein Carla van Doorn Bert Roosendaal Frits K de Graaf Bauke Oudega |
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| Institution: | Department of Microbiology, Biological Laboratory, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands |
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| Abstract: | 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 Mr values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 108 M?1 and 1.0 × 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 Mr of 77 345. |
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| Keywords: | (Bacteriocin iron-chelator primary structure signal sequence binding constants) |
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