The Fc fragments were prepared by papain digestion from human IgG1. The extracellular regions of human FcγRIIIb was expressed using Escherichia coli strain BL21(DE3) as inclusion body. For preparation of deuterated proteins, the bacterial cells were grown in an M9 minimal medium containing 2 g/l of glucose as a 1–3 mixture of isotopically natural and fully deuterated glucose (1,2,3,4,5,6,6-D7, 98%, Cambridge Isotope Laboratories Inc.), along with a 25:75 ratio of H2O and D2O as previously described [6] (link). The inclusion body was suspended in 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl. After sonication, the homogenate was centrifuged at 8000g for 15 min at 4 °C and the precipitate was denatured by 8 M urea. The denatured protein solution was diluted with refolding buffer (50 mM Tris-HCl, pH 8.0, 0.5 M arginine, 5 mM CaCl2, 5 mM reduced form of glutathione, and 0.5 mM oxidized form of glutathione). After two days of incubation at 4 °C, the diluted protein solution was applied to cOmplete His-Tag Purification Resin (Roche) and further purified on the HiLoad 26/60 Superdex 75 pg column (GE Healthcare).
Hiload 26 60 superdex 75 pg column
Manufactured by GE Healthcare
The HiLoad 26/60 Superdex 75 pg column is a size exclusion chromatography column designed for the purification of proteins, peptides, and other biomolecules. It features a matrix of cross-linked agarose and dextran, which allows for the separation of molecules based on their size and molecular weight.
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3 protocols using hiload 26 60 superdex 75 pg column
1
Production and Purification of Deuterated FcγRIIIb
2
Purification of Codon-Optimized AtBIL1/BZR1
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Codon-optimized Arabidopsis thaliana BIL1/BZR1 (21A–104R) was cloned into pGEX-6P-3 (GE Healthcare) with an N-terminal glutathione S-transferase (GST) tag and a human rhinovirus (HRV) 3C protease cleavage site. Isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced overexpression was performed for 2 h at 37 °C. Cells were harvested by centrifugation at 5000 rpm for 15 min and stored at − 80 °C until use. The harvested cells containing GST-fused AtBIL1/BZR1 were resuspended in buffer A (20 mM Tris–HCl at pH 7.5, 1.0 M NaCl, 1 mM DTT and 5% glycerol) and were then lysed by sonication. The cell debris was removed by centrifugation at 40,000 × g for 30 min. The supernatant fractions were then applied to Glutathione Sepharose 4B resin (GE Healthcare). After washing with buffer A, the HRV 3C protease was added to remove the GST tag, and the unfused protein was then eluted with buffer A. The eluate of unfused AtBIL1/BZR1 was concentrated with a Vivaspin 15R device (10,000 MWCO Hydrosart, Sartorius) and further purified by loading onto a HiLoad 26/60 Superdex 75 pg column (GE Healthcare) against buffer B (20 mM Tris–HCl at pH 7.5, 0.5 M NaCl, 1 mM DTT and 5% glycerol). The purified protein was concentrated to 1.0 mM in preparation for cocrystallization with DNA.
3
Production and Purification of Soluble HLA-G2
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The gene encoding ectodomain of HLA-G2 (α1-α3 domains, Gly1-Trp182), which is identical for its soluble form, HLA-G6, was ligated into a pGMT7 plasmid vector. The HLA-G2 ectodomain was expressed as inclusion bodies in BL21(DE3)pLysS (Merck Millipore) for in vitro experiments and ClearColi ® BL21(DE3) competent cells (Lucigen) for in vivo experiments. The HLA-G2 inclusion bodies were refolded by a dilution method for three days and purified by size exclusion chromatography (SEC) using a HiLoad 26/60 Superdex 75 pg column (GE Healthcare) with SEC buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl). For in vivo analyses, the buffer was exchanged by dialysis using phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 and 1.5 mM KH 2 PO 4 ). Both HLA-G2 and PBS (control for in vivo analysis) prior to injection in mice were treated with a Detoxi-gel endotoxin removing column (Thermo Fisher Scientific).
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