| The inclusion body pellets were individually washed with 1 ml of 100 mM Tris-HCl, pH 8.5, 1 M urea, 1.0% Triton X-100, and again with 100 mM Tris-HCl, pH 8.5, 2 M urea, 2.0% Triton X-100. The pellets were resuspended in 1 ml of dH2O and transferred to a preweighed 30 ml Corex centrifuge tube. The sample was centrifuged at 15000×g for 5 min at 4°C, and the pellet was resuspended in 10 ml/g (wet wt. pellet) of 70% formic acid. Cyanogen bromide was added to a final concentration of 400 mM and the sample was incubated at room temperature in the dark for 16 h. The reaction was stopped by transferring the sample to a round bottom flask and removing the solvent by rotary evaporation at 50°C. The residue was resuspended in 20 ml/g (wet wt. pellet) of dH2O, shell frozen in a dry ice ethanol bath, and then lyophilized. The lyophilized protein was dissolved in 20 ml/g (wet wt. pellet) of 500 mM Tris-HCl, pH 8.2, 7 M urea. Oxidative sulfitolysis was performed by adding sodium sulfite and sodium tetrathionate to final concentrations of 100 and 10 mM, respectively, and incubating at room temperature for 3 h. This reaction was stopped by freezing on dry ice.
The S-sulfonated material was applied to a 2 ml bed of Sephadex G-25 equilibrated in 20 mM Tris-HCl, pH 8.2, 7 M urea, and then washed with 9 vols. of 7 M urea. The collected fraction was applied to a Pharmacia Mono Q HR 5/5 column equilibrated in 20 mM Tris-HCl, pH 8.2, 7 M urea at a flow rate of 1 ml/min. A linear gradient leading to a final concentration of 0.5 M NaCl was used to elute the bound material. 2 min (2 ml) fractions were collected during the gradient, and protein concentration in each fraction was determined. Purity and molecular mass of fractions were estimated by Tricine SDS-PAGE. Appropriate fractions were pooled and applied to a 1.6?0 cm column of Sephadex G-25 (superfine) equilibrated in 5 mM ammonium acetate pH 6.8. The sample was collected based on UV absorbance and freeze-dried. The partially purified S-sulfonated material was resuspended in 50 mM glycine/NaOH, pH 10.5 at a final concentration of 2 mg/ml. beta-mercaptoethanol was added at a ratio of 1.5 mol per mol of cysteine S-sulfonate and the sample was stirred at 4°C in an open container for 16 h. |