The nsp1 segment of SARS-CoV-2 was subcloned into 6X His-SMT3 fusion T7 expression vector (pE-SUMO; Life Sensors) using primers to amplify residues 10–127 of ORF1a. Expression plasmids were transformed into DE3 Star BL21 Escherichia coli cells and grown in LB media containing 200 ug/mL of kanamycin. The nsp1 cultures were induced at OD600 of 0.7 with 1mM isopropyl β-D-1-thiogalactopyranoside for 48 h at 18 °C. Cells were lysed via sonication in buffer containing 20 mM HEPES, 500 mM NaCl, 5 mM Imidazole, 5 mM DTT and 1.3 mg/ml lysozyme at pH 7.8. Clarified lysates were loaded onto His-tag purification column (Roche) and the protein was eluted in a buffer containing 20 mM HEPES, 500 mM NaCl, 250 mM Imidazole, 2.5 mM DTT at pH 8. The 6X His-SMT3 tag was removed from the N terminus with recombinant SUMO protease. The cleaved sample was then reloaded onto the His-tag purification column to separate the 6X His-SMT3 tag from nsp1. Buffer exchange was performed with size exclusion chromatography using a Hi-Prep 26/60 Sephacryl S-100 HR (GE). The final buffer is composed of 20 mM NaPO4, 200 mM NaCl, 1 mM EDTA, 50 mM Arginine, 50 mM Glutamic Acid, 0.02% Sodium azide at a pH of 6.8.
His tag purification column
Manufactured by Roche
The His-tag purification column is a chromatography column used for the purification of recombinant proteins containing a histidine tag. The column is designed to selectively bind and isolate these tagged proteins from complex mixtures, allowing for efficient separation and concentration of the target protein.
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2 protocols using his tag purification column
1
Expression and Purification of SARS-CoV-2 nsp1 Protein
2
Purification of SARS-CoV-2 nsp1 Protein
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The nsp1 segment of SARS-CoV-2 was subcloned into a 6× His-SMT3 fusion T7 expression vector (pE-SUMO; Life Sensors) using primers to amplify residues 10 to 127 of ORF1a. Expression plasmids were transformed into DE3 Star BL21 Escherichia coli cells and grown in LB medium containing 200 μg/ml of kanamycin. The nsp1 cultures were induced at an optical density at 600 nm (OD600) of 0.7 with 1 mM isopropyl β-d -1-thiogalactopyranoside for 48 h at 18°C. Cells were lysed via sonication in buffer containing 20 mM HEPES, 500 mM NaCl, 5 mM imidazole, 5 mM dithiothreitol (DTT), and 1.3 mg/ml lysozyme at pH 7.8. Clarified lysates were loaded onto a His tag purification column (Roche), and the protein was eluted in a buffer containing 20 mM HEPES, 500 mM NaCl, 250 mM imidazole, and 2.5 mM DTT at pH 8. The 6× His-SMT3 tag was removed from the N terminus with recombinant SUMO protease. The cleaved sample was then reloaded onto the His tag purification column to separate the 6× His-SMT3 tag from nsp1. Buffer exchange was performed with size exclusion chromatography using a Hi-Prep 26/60 Sephacryl S-100 HR (GE). The final buffer is composed of 20 mM NaPO4, 200 mM NaCl, 1 mM EDTA, 50 mM arginine, 50 mM glutamic acid, and 0.02% sodium azide at pH 6.8.
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