The pet26b vector was used to express HLA-A*02:01 (1–275) and β2M (1–100) separately in Rosetta BL21 DE3 E. coli cells. Inclusion bodies containing the separate proteins were dissolved in 8 M urea, 40 mM Tris-HCl pH 8.0, 10 mM EDTA, and 10 mM DTT. For in vitro refolding, the HLA-A*02 heavy chain, β2M, and MMDFFNAQM peptide were mixed in a 1:2:10 molar ratio and diluted into a refolding buffer containing 0.4 M L-arginine-HCl, 100 mM Tris-HCl pH 8.0, 4 mM EDTA, 0.5 mM oxidized glutathione, and 4 mM reduced glutathione. After 72 hours at 4°C, the protein was dialyzed in 10 L of 10 mM Tris-HCl and purified via weak ion exchange using a DEAE cellulose column. The protein elution was purified using size exclusion chromatography on a Superdex 200 column and ion-exchange chromatography on a 5/50 Mono Q column (GE Healthcare). Protein was biotinylated overnight with birA ligase, 100 uM biotin, 40 mM Bicine pH 8.3, 10 mM ATP, and 10 mM Magnesium Acetate at 4 °C after buffer-exchange to 1X HBS pH 7.2 in a 30 kDa filter (Millipore) before being run on a size exclusion Superdex 200 column.
Superdex 200 column
Manufactured by Merck Group
Sourced in United States, France
The Superdex 200 column is a size exclusion chromatography (SEC) column used for the separation and purification of biomolecules based on their size and molecular weight. It is designed to provide high-resolution separation of proteins, peptides, and other macromolecules.
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5 protocols using superdex 200 column
1
Recombinant HLA-A*02:01 Production
2
Interaction between YdfD and IspG
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Pull-down assays were performed to assess the interaction between YdfD and IspG. YdfD and its various mutants (Table S1 ) were attached to GST for the identification of YdfD–IspG interactions. For GST-YdfD pulled-down IspG-His, E. coli BL21(DE3) cells were cotransformed by pGEX-6P-1-ydfD and pET28a-ispG-his and induced by IPTG. After bacterial cells were harvested, they were lysed and centrifuged and the supernatants were incubated with the GST beads. Finally, the protein sample beads were directly subjected to SDS-PAGE and Western blotting (diluted 1:10) after 3 washes using wash buffer and heat denaturation (98 °C for 10 min). Other samples were prepared by the same method. To prepare large amounts of protein, MBP tag was fused to the N-terminus of YdfD. The complex of MBP-YdfD and IspG was purified via amylose resin; after the resin treatment with wash buffer, the protein was eluted by wash buffer containing 20 mM maltose. IspG protein was purified using His-tag by Ni-NTA resin and eluted by wash buffer containing 300 mM imidazole. Finally, the elution was concentrated by a centrifugal filter (Millipore, Billerica, MA, USA, 10 kD) to 1 mL and loaded onto a Superdex 200 column for SEC analysis. SEC runs were conducted at 4 °C at a flow rate of 1.0 mL/min using a buffer containing 20 mM Tris, 150 mM NaCl, and 0.5 mM DTT at pH = 8.0.
3
Purification of Bacterial Protease
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To obtain a clear supernatant, on the third day of growth at 30 °C, the bacterial culture was filtered and centrifuged at 9000×g for 10 min. The production medium contained (%, m/V) shrimp shell powder 2.5 and fructose 0.5, as the optimum nitrogen and carbon source, respectively, and NaCl 4 (pH=9). The protease was purified in a two-step procedure consisting of ammonium sulphate precipitation (35–85% saturation) and molecular sieve chromatography. All protein purification stages were performed at 4 °C. Fractionation with ammonium sulphate was performed by salting out the enzyme preparation to 35% of saturation in the first, and to 85% of saturation in the second step. To dissolve the pellet, 50 mM Tris-HCl buffer, pH=8.8, with 2 M NaCl was used and the solution was dialyzed overnight against the same buffer. Prior to the enzyme purification, a Superdex 200 column (Sigma-Aldrich) was equilibrated with 50 mM Tris-HCl, pH=8.8, containing 2 M NaCl. Then, the 10-fold concentrated preparation was applied on the column. The active fractions were used to characterize the enzyme.
4
Purification of Typhoid Toxins PltB and PltC
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Both typhoid toxins were purified according to a previously established protocol6 (link). Briefly, pltB, pltA and cdtB-6xHis (PltB-typhoid toxin) or pltC, pltA and cdtB-6xHis (PltC-typhoid toxin) were cloned into pET28a(+) vector (Novagen). E. coli strains carrying these expression vectors were grown to OD600 ~0.8, at which time 250 μM IPTG was added to induce the expression of the toxin genes and the cultures were grown overnight at 30 °C. Bacterial cells were pelleted by centrifugation and lysed. Crude lysates were affinity purified using Nickel resin (Qiagen), followed by cation exchange chromatography using a Mono S column (Sigma-Aldrich) and finally gel filtration using a Superdex-200 column (Sigma-Aldrich). The final fractions were analyzed by SDS–PAGE to confirm purity.
5
Synthesis and Characterization of HCMV Peptide-HLA Complexes
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Nine-mers UL4015-23 peptides from 11 different HCMV strains (VMAPRTLIL, VMAPRTLLL, VMAPRTLVL, VMAPRTVLL, VMAPRSLIL, VMAPRSLLL, VMTPRTLVL, VMAPQSLLL, VTAPRTLLL, VTAPRTVLL, VMAPRALLL) and the UL83 pp65495-503 peptide (NLVPMVATV) were synthesized (purity>95%) and purchased from Proteogenix SAS (Schiltigheim, France). HLA-E*01:01/UL4015-23 (HLA-EUL40) and HLA-A*02:01/pp65495-503 (HLA-A*02pp65) complexes were generated as described previously [57 (link)]. Recombinant HLA proteins were produced in E.coli and refolded with 15μg/mL of each UL4015-23 peptide for HLA-E-monomers or pp65495-503 peptide for HLA-A*02-monomers. Next, HLA-monomers were biotinylated for 4h at 30°C with 6μg/mL BirA (Immunotech, Marseille, France), purified and tetramerized with BV421- or APC-labelled streptavidin (BD Biosciences, Le Pont de Claix, France). Tetramerization was confirmed by gel filtration chromatography (Superdex 200 column, Sigma-Aldrich, Saint-Quentin Fallavier, France).
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