The extracellular domains of MHC Class II (DRA∗01:01/DRB1∗04:01) α and β chains were designed in pcDNA3.4 TOPO, with both chains containing C-terminal Tobacco Etch Virus (TEV)-cleavable Fos/Jun zippers to promote dimerization and 6xHis tags for purification purposes.78 (link) The MHC Class II molecule was expressed with the Influenza Hemagglutinin (HA) peptide covalently attached via a flexible linker to the N terminus of the β chain to promote proper folding of the α/β dimer. HLA-DR α and β chain plasmids were co-transfected in a 1:1 ratio (50 μg total per 200 mL of cell culture) into HEK 293S (GnT I-/-) cells. Recombinant MHC Class II was purified by affinity chromatography via a HisTrap Ni-NTA column (Cytiva) and eluted using 20 mM Tris, pH 8.0, 500 mM imidazole. Subsequent size exclusion chromatography was performed in TBS, pH 8.0 on a Superdex 200 Increase 10/300 GL (Cytiva). Purified MHC Class II was subjected to overnight treatment with EndoH and TEV proteases at ratios of 5:1 and 20:1, respectively, for deglycosylation and cleavage of the Fos/Jun zipper. A second round of affinity and size exclusion purifications was performed on cleaved MHC Class II before complexation with c44H10 Fab for crystallization trials.
Histrap ni nta column
Manufactured by Cytiva
The HisTrap Ni-NTA column is a pre-packed affinity chromatography column designed for the purification of His-tagged recombinant proteins. The column contains Ni Sepharose resin, which binds to the His-tag on the target protein, allowing for selective capture and purification.
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3 protocols using histrap ni nta column
1
Recombinant MHC Class II Production
2
Purification of His-tagged RccR Protein
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The pET28a-RccR-his plasmid was transformed into E. coli BL21(DE3), and the cells were grown in LB with 50 μg/ml kanamycin at 37°C until the optical density at 600 nm (OD600) reached 0.6. The isopropyl-β-d -thiogalactopyranoside was supplemented at a final concentration of 0.25 mM to induce the protein expression overnight at 16°C. Cells were harvested by centrifugation and the pellet was resuspended in buffer A (10 mM Tris–HCl, pH 7.5, 1 M NaCl and 1 mM DTT), and then the suspension was disrupted by sonication and clarified by centrifugation. The supernatant was filtered and then loaded onto a 5-ml HisTrap Ni-NTA column (Cytiva). The column was washed with buffer A containing 62.5 mM imidazole for unbound proteins, and the His-tagged RccR protein was eluted with buffer A containing 500 mM imidazole. The eluted protein was concentrated to 2 ml and loaded onto a HiLoad 16/600 Superdex 200pg column (Cytiva) for further elution with buffer A. The purified protein was concentrated for subsequent experiments.
3
SARS-CoV-2 RBD and hACE2 Protein Purification
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FreeStyle 293-F cells were transfected as described above with 50 μg of plasmid DNA encoding the SARS-CoV-2 RBD or human ACE2 (hACE2) per 200 mL of cell culture. Recombinant proteins were purified by affinity chromatography via a HisTrap Ni-NTA column (Cytiva) and eluted using 20 mM Tris pH 8.0, 500 mM imidazole buffer. Subsequent size exclusion chromatography was performed in 20 mM Tris, pH 8.0, 150 mM NaCl on a Superdex 200 Increase column (Cytiva).
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