| Expression of PvRII in E. coli-- Luria broth containing kanamycin (50 µg/ml) was inoculated with E. coli BL21(DE3)pVPET1 and cultured overnight at 37 °C. Fresh Luria broth containing kanamycin (25 µg/ml) was inoculated with the overnight culture at a dilution of 1:50 and cultured at 37 °C to an A600 nm of 0.6-0.8. Expression of PvRII was induced by adding isopropyl-1-thio--galactopyranoside to the culture at a final concentration of 1 mM. Induced cultures were allowed to grow for 4 h at 37 °C.
Isolation of Inclusion Bodies and Purification of Recombinant PvRII by Metal Affinity Chromatography under Denaturing Conditions-- AFter induction, E. coli cells were harvested by centrifugation, washed in chilled wash buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl), resuspended in chilled lysis buffer (10 mM Tris, pH 8.0, 5 mM benzamidine-HCl, 2 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 100 mM NaCl, 200 µg/ml lysozyme), and lysed by sonication. Inclusion bodies were collected by centrifugation of lysed cells at 4 °C, solubilized in 10 mM Tris buffer, pH 8.0, containing 6 M guanidine hydrochloride (GdnHCl), and treated with 10 mM dithiothreitol (DTT) for 2 h at 37 °C. After removal of DTT by ultrafiltration using filters with 10-kDa cutoff, recombinant PvRII was purified from solubilized inclusion bodies by metal affinity chromatography under denaturing conditions using a nickel nitrilotriacetic acid column as described by the manufacturer (Qiagen). Solubilized inclusion bodies were loaded on a nickel nitrilotriacetic acid column previously equilibrated with equilibration buffer (10 mM Tris, pH 8.0, 100 mM NaH2PO4, 6 M GdnHCl). The column was washed with equilibration buffer at pH 6.3, and bound protein was eluted using a pH gradient starting at pH 6.3 and ending at pH 4.3. The final concentration of the purified protein was adjusted to ~4.5 mg/ml with equilibration buffer.
Refolding PvRII by Rapid Dilution-- Purified PvRII was refolded by 100-fold dilution in refolding buffer containing 50 mM phosphate buffer, pH 7.2, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 1 M urea, and 0.5 M arginine so that the final protein concentration was ~45 µg/ml. Refolding was allowed to proceed at 10 °C for 36 h with stirring. At the end of 36 h, the refolding solution was dialyzed for 48 h against dialysis buffer (50 mM phosphate buffer, pH 6.5, 1 M urea) to remove arginine before proceeding with purification by ion-exchange chromatography.
Purification of Refolded PvRII by Ion Exchange and Gel Filtration Chromatography-- After removal of arginine by dialysis, the refolded protein was loaded on an SP-Sepharose column equilibrated with 50 mM phosphate buffer, pH 6.5. The bound protein was eluted with a linear gradient of NaCl (100 mM NaCl to 1.5 M NaCl). Fractions containing refolded PvRII were pooled, and PvRII was further purified by gel filtration chromatography using a Superdex 75 column (Amersham Pharmacia Biotech). For gel filtration chromatography, 50 mM phosphate buffer, pH 7.2, containing 200 mM NaCl was used
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