SARS-CoV PLpro, UCH-L3, and MERS-PLpro were obtained as described earlier (14 (link), 25 (link)). SARS-CoV-2 PLpro transformed cells were grown in LB broth at 37°C with shaking until the optical density at 600 nm reached 1.5. Isopropyl-β-d -thiogalactopyranoside (0.1 mM) and ZnSO4 (0.1 mM) were added to induce protein expression overnight at 18°C. Cell pellet was resuspended in lysis buffer [20 mM tris-Cl (pH 8.0), 350 mM NaCl, and 2 mM β-mercaptoethanol) and lysed using sonication. The lysate was cleared by centrifugation at 35,000g for 30 min at 4°C. The lysate was passed onto Glutathione Sepharose 4B (GE) followed by washing with lysis buffer. The GST-tagged PLpro was eluted in lysis buffer supplemented with 20 mM reduced glutathione (pH 8.0). The fusion protein was cleaved using GST-PreScission protease at 4°C overnight followed with desalting and passing through fresh glutathione beads to remove cleaved GST and PreScission protease. The sample was further purified using Superdex 200-pg size-exclusion columns (GE) equilibrated with 20 mM tris-Cl (pH 8.0), 40 mM NaCl, and 2 mM dithiothreitol (DTT). The purified protein was then concentrated to ~10 mg/ml and snap-frozen in liquid nitrogen for later use.
Superdex 200 pg size exclusion column
Manufactured by GE Healthcare
Superdex 200-pg size-exclusion columns are laboratory equipment used for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. These columns utilize a porous resin material to fractionate samples based on their molecular size during liquid chromatography. The size-exclusion mechanism allows larger molecules to elute first, while smaller molecules are retained within the porous matrix.
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4 protocols using superdex 200 pg size exclusion column
1
Purification of SARS-CoV-2 PLpro Enzyme
2
SARS-CoV-2 PLpro Protein Purification
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SARS-CoV-2 PLpro was prepared as described26 (link). In brief, pGEX6P-1-SARS-CoV-2PLpro was transformed into BL21 (DE3) codon-plus E. coli cells and induced with 0.1 mM IPTG and 0.1 mM ZnSO4 at 18 °C overnight. GST-fusion SARS-CoV-2 PLpro was purified using a standard protocol. The fusion protein was cleaved using GST-PreScission protease at 4 °C overnight followed with desalting and passing through fresh glutathione beads to remove cleaved GST and GST-PreScission protease. The sample was further purified using Superdex 200 pg size-exclusion columns (GE) equilibrated with 20 mM Tris–Cl pH 8.0, 40 mM NaCl and 2 mM DTT. The peak fractions were pooled and concentrated to ~ 10 mg/mL and snap frozen in liquid nitrogen for later use.
3
Purification of Heme Oxygenase Enzyme
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HemO was purified as previously reported with slight modification (12 (link)). HemO lysate was applied to a Q-Sepharose Fast Flow column (2.5 × 6 cm) (GE Life Sciences) equilibrated with 20 mM Tris (pH 8.0 at 4 °C) and 100 mM NaCl. Protein was eluted with a 20 mM Tris (pH 8.0 at 4 °C) and 100 to 500 mM NaCl gradient. Peak protein fractions were determined via SDS-PAGE and were pooled, concentrated, and dialyzed against 20 mM Tris buffer (pH 8.0) and 100 mM NaCl at 4 °C. The protein (5–6 ml) was further purified by FPLC over a 26/60 Superdex 200 pg size exclusion column (GE Life Sciences) equilibrated with 20 mM Tris (pH 8.0) containing 100 mM NaCl. Peak fractions as judged by the A280 were subjected to SDS-PAGE, and the pure fractions were pooled, concentrated, (10 mg/ml), and stored at −80 °C until further use.
4
HemO Protein Purification by Ion Exchange and Size Exclusion Chromatography
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HemO was purified as previously reported with slight modification (12) . HemO lysate was applied to a Q-Sepharose Fast Flow column (2.5 cm × 6 cm) (GE Life Sciences) equilibrated with 20 mM Tris (pH 8.0 at 4°C), 100 mM NaCl. Protein was eluted with a 20 mM Tris (pH 8.0 at 4°C), 100-500 mM NaCl gradient. Peak protein fractions were determined SDS-PAGE and were pooled, concentrated and dialyzed against 20 mM Tris buffer (pH 8) 100 mM NaCl at 4 o C. The protein (5-6 ml) was further purified by FPLC over a 26/60 Superdex 200 pg size exclusion column (GE Life Sciences) equilibrated with 20 mM Tris (pH 8.0) containing 100 mM NaCl. Peak fractions as judged by the A280 were subjected to SDS-PAGE and the pure fractions pooled, concentrated (10 mg/ml) and stored at -80 o C until further use.
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