Equine myoglobin

Proteins were purified from soluble fractions in one of two ways. For chimeras containing OspA, the supernatant containing the 6 × -His-tagged protein of interest was purified using an ÄKTA Explorer FPLC system (GE Healthcare) over a Ni²⁺ Sepharose High-Performance HisTrap HP column (GE Healthcare). For chimeras containing ΔspMBP, purification was performed according to the manufacturer's protocol supplied with pMAL vectors (New England Biolabs). SEC was performed on all 6 × -His-tagged and ΔspMBP-tagged purified proteins. Standards used to calibrate the SEC column were a lyophilized mix of thyroglobulin, bovine γ-globulin, chicken ovalbumin, equine myoglobin and vitamin B12, MW 1,350-670,000, pI 4.5-6.9 (Bio-Rad). Proteins were stored at a final concentration of 1 mg ml⁻¹ in SEC buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA pH 8.0) at 4 °C. Expression and purification of EmrE was according to standard protocols43 (link).

The triple mutant and wild type EphA4 Fc proteins were injected onto a TSK-GEL SWXL guard column (6.0 mm ID × 400 mm, 7-μm particles) and subsequently eluted onto a TSK-GEL G3000SWXL HPLC column (7.8 mm ID × 300 mm, 5-μm particles) using an Agilent 1200 chromatography system. The flow rate was 0.8 ml/min and the mobile phase contained 100 mM sodium phosphate (pH 6.8) and 200 mM NaCl. Eluted proteins were monitored by UV absorption (280 nm wavelength). The following standards were used: thyroglobin (670 kDa), bovine γ-globin (158 kDa), ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa) (BioRad Laboratories, Hercules, CA, USA).

The catalytic domain of wild-type Rv1625c (Rv1625c-Wt) encompassing residues Met212–Val443 was cloned into pPRO-EX-HT vector to generate a hexahistidine at the N-terminus. The F363R mutant of Rv1625c (Rv1625c-F363R) was generated by performing site-directed mutagenesis as described previously (Shenoy et al., 2003 ▶ ). Expression and purification of the protein was carried out as described previously (Ketkar et al., 2004 ▶ ). The protein obtained after Ni–NTA purification was further purified and its oligomeric state was assessed using gel filtration. Gel filtration was performed using a Superose 12 10/300 GL column (Amersham Pharmacia Biotech) equilibrated with buffer consisting of 20 mM Tris pH 7.4, 10%(v/v) glycerol, 5 mM β-mercaptoethanol (β-ME), 10 mM NaCl on an ÄKTA fast protein liquid chromatography (FPLC) system at a flow rate of 0.25 ml min⁻¹. 10% glycerol was required to keep the protein in a stable form. The column was calibrated with standard protein molecular-weight markers from Bio-Rad: bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B₁₂ (1.3 kDa).

To verify the oligomeric state and to ensure purified protein is not aggregated, APRT1 and APRT2 were subjected to SEC. Purified APRT1and APRT2 were concentrated to 13.8 mg mL^-1 and 10.8 mg mL^-1 respectively. 200 μL of concentrated APRT1 or APRT2 were applied to a GE Superdex 200 Increase 10/300 GL SEC column using an ÄKTA pure (GE Healthcare). Proteins were eluted off with 1.2 CV of SEC Buffer: 50 mM HEPES (pH 7.8), 300 mM NaCl, 50 mM L-arginine, 50 mM L-glutamate. Prior to sample application the column was calibrated with lyophilized standards ranging from 1,350 to 670,000 Da molecular weight, containing a mixture of thyroglobulin, bovine γ-globulin, chicken ovalbumin, equine myoglobin, and vitamin B12 (BioRad, Cat #1511901) resuspended in SEC Buffer. Samples were compared to the standard to ascertain the M_r (relative molecular weight), which is indicative of the oligomeric state.

Gel filtration analysis of protein was performed at 4 °C using an Sephacryl HiPrep 16/60 S200 HR column (GE Healthcare) on an AKTA Pure chromatography system (GE Healthcare). Protein was dialyzed overnight against running buffer (20 mm Tris-HCl, pH 7.4, 140 mm NaCl, 1 mm DTT). Proteins were incubated on ice for 30 min individually or together and then centrifuged at 12,000 × g for 30 min. Protein loading concentration was ∼75 μm. Columns were run at a flow rate of 0.5 ml/min in running buffer. Thirty μl of each eluted fraction was subjected to SDS-PAGE and visualization with Quick Coomassie stain (Generon). Molecular mass was calibrated with gel filtration standards (Bio-Rad): bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa).