Plasmids for HLA-A*02:01 and β2M were received from the NIH Tetramer Facility and separately transformed into BL21(DE3) cells. Each was expressed in inclusion bodies using auto-induction medium as previously described (64 (link)-66 (link)). Purification of the HLA-A*02:01 and β2M inclusion bodies was achieved with a series of detergent washes followed by solubilization with 8 M urea. Refolding of the HLA-A*02:01, β2M, and mutant p53R175H peptide was performed as previously described (28 (link)). Briefly, solubilized HLA-A*02:01 and β2M were combined in a refolding buffer containing 100 mM Tris pH 8.3, 400 mM l -arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 mM PMSF, and 30 mg of the mutant p53R175H peptide (amino acids 168 to 176, HMTEVVRHC) dissolved in ml of DMSO. The resultant solution was stirred at 4°C for 2 days, with two further additions of HLA-A*02:01 on day 2, concentrated to 10 ml, and purified by size exclusion chromatography on a HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare, 28989334). For incubation with the H2-Fab, purified pHLA-A*02:01 was concentrated to ~1 to 3 mg/ml and stored at −80°C until use.
Hiload 26 60 superdex 75 prep grade column
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The HiLoad 26/60 Superdex 75 Prep grade column is a size exclusion chromatography column designed for the purification of proteins, peptides, and other biomolecules. It features a pre-packed matrix of cross-linked agarose and dextran that separates molecules based on their size and hydrodynamic radius.
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Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nap 5 column, by GE Healthcare (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Superdex 200 10 30 column, by GE Healthcare (1 mentions)
Talon chromatography, by Takara Bio (1 mentions)
Crystal gryphon robot, by Art Robbins Instruments (1 mentions)
6 protocols using hiload 26 60 superdex 75 prep grade column
1
Recombinant HLA-A*02:01 Refolding Protocol
2
Azide Conversion of eGFP Protein
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A construct of eGFP in the pOPIN_J vector was a gift from Owen Burbidge (University of Cambridge). The protein was expressed in E. coli cells and purified using nickel-nitrilotriacetic acid (Ni–NTA) beads (QIAGEN, Manchester, UK) followed by size-exclusion chromatography on a HiLoad 26/60 Superdex 75 prep-grade column (GE Healthcare, Little Chalfont, UK). Purified protein was flash-frozen and stored at −80 °C.
The N-terminal amine of the eGFP was converted to an azide using the protocol of Schoffelen et al. [39 (link)]. This method reliably converts N-terminal amines to azides without modifying lysine residues. The protein was exchanged into 50 mM Clark and Lubs buffer at pH 8.9 using a NAP-5 column (GE Healthcare) to give a 71.8 µM solution. 240 µL of this was added to 227 µL of 50 mM Clark & Lubs buffer at pH 8.9 and 33 µL of a 2 mg/mL solution of imidazolyl-1-sulfonyl azide (synthesized by Yu Heng Lau, University of Cambridge, according to a literature procedure [40 (link)]). The mixture was shaken at 37 °C overnight, and the extent of diazotization checked by mass spectrometry.
The N-terminal amine of the eGFP was converted to an azide using the protocol of Schoffelen et al. [39 (link)]. This method reliably converts N-terminal amines to azides without modifying lysine residues. The protein was exchanged into 50 mM Clark and Lubs buffer at pH 8.9 using a NAP-5 column (GE Healthcare) to give a 71.8 µM solution. 240 µL of this was added to 227 µL of 50 mM Clark & Lubs buffer at pH 8.9 and 33 µL of a 2 mg/mL solution of imidazolyl-1-sulfonyl azide (synthesized by Yu Heng Lau, University of Cambridge, according to a literature procedure [40 (link)]). The mixture was shaken at 37 °C overnight, and the extent of diazotization checked by mass spectrometry.
3
Synthesis of Lys48-linked Ubiquitin Chains
4
Purification and Reconstitution of Iron-Sulfur Enzymes
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WT Bsub_RlmN and evolved variants were expressed, purified and reconstituted for their iron-sulfur clusters using modified versions of previously published protocols (18 (link),34 (link),35 (link)). Briefly, enzymes were overexpressed and purified by Talon chromatography (Clontech). After chemical reconstitution of the iron-sulfur cluster, proteins were further purified by FPLC either on a Superdex 200 10/30 column or on a HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare Life Sciences) using 10 mM HEPES (pH 7.5) buffer containing 500 mM NaCl, 10% glycerol and 5 mM DTT. The fractions containing protein were combined and concentrated before being stored at −80°C.
5
Fab:fHbp Complex Crystallography
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After overnight incubation at 4°C, the Fab:fHbp complex was purified by size exclusion chromatography using a pre-packed HiLoad 26/60 column Superdex 75 prep grade (GE Healthcare) following manufacturer protocol. After removal of monomeric proteins, the Fab:fHbp dimeric complex was concentrated to 25 mg/ml in 50mM Tris-HCl and was screened in over 800 different crystallization screening experiments. Using a Crystal Gryphon robot (Art Robbins Instruments), each experiment was prepared using 200nl reservoir and 200nl protein sample. The best crystal was grown in buffer containing 0.1M HEPES with 20% w/v jeff ED-2001 as precipitant at pH 6.5. Crystals were soaked in the original mother liquor supplemented with 15% ethylene glycol prior to cryo-cooling in liquid nitrogen.
6
Expression and Purification of fHbp Variants
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fHbp variants were expressed as recombinant protein with a C-terminal hexahistidine tag in E. coli BL21 (DE3). Cultures were grown in HTMC medium and protein expression was induced by IPTG 1 mM for 24 h at 25°C. E. coli cells were lysed by cell lytic express (MilliporeSigma, Burlington, MA, USA) as in the manufacturer’s instruction and centrifuged at 9000 rpm for 30 min. The soluble fraction was then filtered by 0.22-μl filter (MilliporeSigma) to remove cell debris and purified by affinity chromatography. Protein concentration was determined by NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and its purity was assessed by SDS-PAGE on a 4–12% Bis-Tris Gel after Problue Safe Stain (Giotto Biotech, Florence, Italy). When necessary, a second purification step of ion-exchange chromatography was performed to remove all the impurities using a prepacked HiLoad 26/60 Column Superdex 75 Prep Grade (GE Healthcare, Waukesha, WI, USA), according to the manufacturer’s protocol.
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