Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12.
A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D003087:Colicins / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D005291:Ferrichrome / Q000031:analogs & derivatives / Q000378:metabolism; D005960:Glucosides; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010957:Plasmids; D013604:T-Phages / Q000378:metabolism; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003087:Colicins; D005960:Glucosides; D005291:Ferrichrome; C015657:albomycin; C018619:octyl-beta-D-glucoside; D004492:Edetic Acid; D004262:DNA Restriction Enzymes