The method developed by Fernandes et al. (40) for isotopic labeling of c-type multiheme cytochromes was also tested. The cells were first grown in 1 L LB medium supplemented with 50 mg/L dALA, 35 mg/L chloramphenicol, and 100 mg/L ampicillin at 30°C and 180 rpm. After achieving an OD600nm ╛ 1.5, the cells were harvested by centrifugation at 6400× g for 30 min. The cell pellet was washed twice with a salt solution containing 15 g/L KH2PO4, 38 g/L Na2HPO4·H2O, and 2.5 g/L NaCl, in order to remove nutrients left behind by the rich medium. The cells were subsequently resuspended in 250 mL minimal medium containing 5 g/L NH4Cl, 0.5 g/L MgSO4, 0.01 g/L CaCl2, 1 mg/L MnCl2·4H2O, 2.8 mg/L FeSO4·7H2O, 1× BME vitamins solution, 4 g/L glucose, or 7 mL/L 50% glycerol stock solution as carbon source, supplemented with 35 mg/L chloramphenicol, 100 mg/L ampicillin, and 1 mM dALA. Cells were allowed to recover for a period of 1 h before inducing protein expression. Different concentrations of IPTG, ranging from 0 to 1 mM, and also different temperatures (25°, 30°, or 37°C) were used.
The auto-induction method developed by Studier (41) that had previously been used to produce heme proteins in E. coli (42) was also tested. The cells were first grown overnight in LB medium supplemented with 50 mg/L dALA, 35 mg/L chloramphenicol, and 100 mg/L ampicillin at 30°C and 180 rpm. One percent of this culture served as inoculum. The auto-induction medium was made by adding 50 mL stock solution 20× NPS [1 M Na2HPO4, 1 M KH2PO4, 500 mM (NH4)2SO4], 20 mL stock solution 50× 5052 (25% glycerol, 2.5% glucose, 10% α-lactose monohydrate) to 1 L LB or TB medium supplemented with 1 mM MgSO4, 0.1 mM FeSO4, 35 mg/L chloramphenicol, and 100 mg/L ampicillin. The optimal dALA concentration was determined, by testing concentrations varying from 20 to 200 mg/L. Different temperatures (25°, 30°, and 37°C) were also tested in order to optimize protein expression.
In order to test different induction periods, aliquots (1 mL) were taken each hour from each different culture condition and centrifuged to collect the cell pellet. The pellets were subsequently lysed using BugBuster protein extraction reagent. The supernatants were run on a SDS-PAGE, and the gels were stained using the heme staining procedure developed by Francis & Becker (43).
The gels were visualized and the stained protein bands were compared and analyzed for their intensity using the ImageJ v1.44 program (http://rsbweb.nih.gov/ij/index.html).
All growth conditions and analyses were performed in triplicate.Protein production and purification
The auto-induction method previously described was used to overexpress the mutant STC(D2N) multiheme cytochrome in E. coli strain LS543-STC(D2N). The auto-induction medium was supplemented with 35 mg/L chloramphenicol, 100 mg/L ampicillin, 50 mg/L unlabeled dALA to produce unlabeled hemes, or 50 mg/L 1,2-13C-labeled dALA (35) to produce specific isotopically labeled hemes.
The cells were grown for 27 h at 30°C and 180 rpm in conical flasks filled with one-fifth the total volume. Expression of STC(D2N) was induced spontaneously by depletion of the glucose present in the medium. There was a clear change in the medium's color to orange. The bacterial cells were harvested by centrifugation at 4°C and at 10,000× g for 15 min. The cell pellet was disrupted by gentle stirring for 20 min at room temperature with 5 mL BugBuster protein extraction reagent per gram of wet cell paste supplemented with a protease inhibitor cocktail. Cell debris were removed by centrifugation at 16,000× g for 20 min at 4°C. The supernatant containing the soluble extract was loaded directly onto a Q-Sepharose column (GE Healthcare, Little Chalfont, UK), equilibrated previously with 10 mM Tris buffer, pH 7.6. A salt gradient from 0 to 1 M NaCl in 10 mM Tris, pH 7.6, was applied and the fraction containing STC(D2N) was eluted at 200 mM. The final purification step was performed on a hydroxylapatite (HTP) column (Bio-Rad Laboratories, Hercules, CA, USA), preequilibrated with 10 mM phosphate buffer, pH 7.6. The fraction containing STC(D2N) did not bind to the column and was eluted in the washout volume.
The chromatographic fractions were routinely analyzed by SDS-PAGE and UV-visible spectroscopy to select those containing STC(D2N). The purity of the protein was confirmed by a single band on SDS-PAGE. Pure samples present an absorption ratio A408nm/A280nm of approximately 4.5 in the UV-visible spectrum.