Protein was eluted with an imidazole step gradient and was dialyzed extensively against 100 mM sodium phosphate, pH 7.9. Anti-TonB antibodies. TonB variants with altered N termini retain the ability to bind to outer membrane receptors Tolnaftate (22, 29, 35), point mutations affecting this predicted -helical transmembrane domain disrupt interaction with the cytoplasmic membrane protein ExbB and abolish ferric siderophore transport and killing by TonB-dependent phages and protein antibiotics termed colicins (29, 32). Mutations in ExbB or ExbD or overexpression of TonB without concomitant overexpression of ExbB or ExbD increased the rate of degradation of TonB and reduced TonB-dependent receptor activity (1, 18, 53). Such susceptibility to endogenous proteolysis has impeded isolation of TonB from TonB lacking the N-terminal cytoplasmic membrane anchor. Here we describe purification of this hexahistidine-tagged TonB variant (H6-TonB), show that it retains affinity for the TonB-dependent receptors FhuA and FepA, and demonstrate that it responds to the ligand occupation status of these receptors. The stability of H6-TonB and its ability to interact with TonB-dependent receptors imply that the protein is amenable to further structure-function studies. MATERIALS AND METHODS Bacterial strains and reagents. XL-1 Blue [(F hsdR17 (rK? mK+) ? (((((43), and KP1120(43) were used to evaluate the specificity and titer of polyclonal anti-TonB chicken serum. QIAprep spin miniprep columns, a QIAquick PCR purification kit, and Ni2+-nitrilotriacetate (NTA) agarose resin were purchased from Qiagen (Courtaboeuf, France); plasmid pET-28 was obtained from Novagen (Madison, Wis.). Restriction enzymes, thermostable proofreading DNA polymerase, and T4 DNA ligase were purchased from Eurogentec (Seraing, Belgium). Antihistidine monoclonal antibody, bromochloroindolyl phosphate, nitroblue tetrazolium, phenylmethylsulfonyl fluoride, and formaldehyde ZC3H13 (37% solution in water) were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Protease inhibitor cocktail (Complete) tablets and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc SC) were purchased from Roche Diagnostics (Meylan, France) and Tolnaftate were used as recommended by the manufacturer. Cloning of TonB. The gene (46) was amplified by PCR from 20 ng of chromosomal DNA of XL-1 Blue. The reaction mixture included 1.5 U of the proofreading DNA polymerase and 50 pmol (each) of primers that were designed to incorporate 5 sequence and is preceded by the codon for the C-terminal Gln239 of the TonB sequence). PCR products were trimmed with ER2566, and 500-ml cultures in Luria broth plus kanamycin (30 g/ml) were induced with 0.5 mM isopropyl Tolnaftate -d-thiogalactopyranoside (IPTG). Cells were collected by centrifugation and suspended in 50 ml of 100 mM sodium phosphate, pH 7.9. Protease inhibitor cocktail tablets and phenylmethylsulfonyl fluoride were added, and all steps were carried out at 4C or on ice. Cells were lysed by sonication, and the cleared supernatant was mixed with 5 ml of equilibrated Ni2+-NTA agarose resin in batch for 30 min. Protein was eluted with an imidazole step gradient and was dialyzed extensively against 100 mM sodium phosphate, pH 7.9. Anti-TonB antibodies. White Leghorn hens (Genaxis, Montigny le Bretonneux, France) were immunized subcutaneously at 3-week intervals with 100 g of purified H6-TonB in Freund’s complete adjuvant. Immune serum drawn 2 weeks after the first boost revealed anti-TonB titers of 1 1:15,000 relative to the preimmune control serum in an enzyme-linked immunosorbent assay (ELISA) with purified H6-TonB. The specificity of the anti-TonB immune serum was confirmed by blotting against whole-cell extracts of KP1060 (TonB+) and KP1120 (= 8.6 nm; = 8.8 ml), ferritin (= 6.3 nm; = 10.7 ml), catalase (= 5.2 nm; = 11.7 ml), aldolase (= 4.6 nm; = 12.0 ml), bovine serum albumin (= 3.5 nm; = 12.5 ml), ovalbumin (= 2.8 nm; = 13.4 ml), chymotrypsinogen A (= 2.1 nm; = 14.9 ml), and RNase A (= 1.75 nm; = 15.5.