The heavy chains and light chains of 54–4H03 and 54–1G05 were cloned into a phCMV3 vector. The heavy chain and light chain plasmids were co-transfected into ExpiCHO cells (Thermo Fisher Scientific) at 2:1 molar ratio (heavy to light) using the Max titer protocol as described by the manufacturer’s instructions for the ExpiCHO Expression System. Fabs were purified from the supernatant by a 5 mL HiTrap Protein G HP antibody purification column ÄKTAxpress (GE Healthcare) and subsequently by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3. For crystallization of the apo forms, Fabs were further buffer exchanged into 10 mM Tris pH 8.0, 50 mM NaCl, and 0.02% NaN3, and concentrated to 10 mg mL-1.
Hiload 16 90 superdex 200 column
Manufactured by GE Healthcare
The Hiload 16/90 Superdex 200 column is a size exclusion chromatography column used for the separation and purification of biomolecules such as proteins, peptides, and other macromolecules. It features a prepacked matrix of cross-linked agarose and dextran to enable efficient separation based on the size and shape of the molecules.
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Lab products found in correlation
Fugene hd, by Promega (3 mentions)
Bac to bac system, by Thermo Fisher Scientific (3 mentions)
High five cells, by Thermo Fisher Scientific (3 mentions)
Expicho cells, by Thermo Fisher Scientific (2 mentions)
Hitrap protein g hp antibody purification column ktaxpress, by GE Healthcare (2 mentions)
Hitrap, by Cytiva (2 mentions)
Trypsin, by GE Healthcare (2 mentions)
Trypsin, by New England Biolabs (2 mentions)
Pfuse fc vector, by InvivoGen (1 mentions)
Ni nta superflow, by Qiagen (1 mentions)
Ni sepharose excel histidine tagged protein purification resin, by GE Healthcare (1 mentions)
Centramate cassette, by Pall Corporation (1 mentions)
7 protocols using hiload 16 90 superdex 200 column
1
Fab Protein Expression and Purification
2
Fab Purification and Crystallization Protocol
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The heavy chains and light chains of 54-4H03 and 54-1G05 were cloned into a phCMV3 vector. The heavy chain and light chain plasmids were co-transfected into ExpiCHO cells (Thermo Fisher Scientific) at 2:1 molar ratio (heavy to light) using the Max titer protocol as described by the manufacturer’s instructions for the ExpiCHO Expression System. Fabs were purified from the supernatant by a 5 mL HiTrap Protein G HP antibody purification column ÄKTAxpress (GE Healthcare) and subsequently by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3. For crystallization of the apo forms, Fabs were further buffer exchanged into 10 mM Tris pH 8.0, 50 mM NaCl, and 0.02% NaN3, and concentrated to 10 mg mL-1.
3
Efficient Fab Variant Expression and Purification
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Individual mutants for the validation experiment were constructed using the QuikChange XL Mutagenesis kit (Stratagene, San Diego, CA) according to the manufacturer's instructions. Primers for Quikchange were designed such that they matched 18 bp flanking each side of the mutated region. The nucleotide sequence of the mutated region on the primers was designed to minimize nucleotide mismatch with the WT C05 Fab. For the expression of C05 Fab variants in mammalian cells, both light and heavy chains were cloned into the pFuse vector, which was constructed by removing the Fc-encoding sequence from the pFuse-Fc vector (Invivogen, San Diego, CA). The light chain and heavy chain were transfected into 293T cells in a 2:1 molar ratio. A His6-tag was inserted at the C-terminus of the heavy chain. The supernantants containing the C05 Fab variants were collected 3 days after transfection. Expression of C05 Fab variants in insect cells was performed as described previously for WT C05 Fab21 (link). Purification of C05 Fab variants was performed by Ni-NTA Superflow (Qiagen, Valencia, CA), and subsequently by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare, Pittsburgh, PA) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3.
4
Recombinant HA Purification for Structural Analysis
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Preparation of recombinant HA for nsEM and mass spectrometry was as previously described [78 (link),79 (link)]. The HA ectodomain in H3 numbering (11 to 329 in HA1 and 1–176 in HA2, respectively) was fused with an N-terminal secreting signal peptide and followed by a C-terminal BirA biotinylation site, thrombin cleavage site, T4 trimerization domain, and Hisx6 tag in a phCMV3 vector. Different HA plasmids were transfected into Expi293F cells to express soluble recombinant HAs. Incubation conditions and harvest procedures were performed according to manufacturer’s guide (Thermo Fisher Scientific). HA proteins generated from Expi293F cells were purified by Ni Sepharose excel histidine-tagged protein purification resin (GE Healthcare), buffer exchanged with 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. All HAs were treated with trypsin and purified by size exclusion chromatography (SEC) using a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3.
5
Recombinant Influenza Hemagglutinin Expression
6
SARS-CoV-2 and SARS-CoV Spike Protein RBD Expression and Purification
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The receptor-binding domain (RBD) (residues 319–541) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1 ), and the RBD (residues 306–527) of the SARS-CoV S protein (GenBank: ABF65836.1 ), were cloned into a customized pFastBac vector [47 (link)], and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag [13 (link)]. For each RBD, we further cloned a construct with an AviTag inserted in front of the His6 tag. To express the RBD, a recombinant bacmid DNA was generated using the Bac-to-Bac system (Life Technologies). Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGENE HD (Promega), and subsequently used to infect suspension cultures of High Five cells (Life Technologies) at an MOI of 5 to 10. Infected High Five cells were incubated at 28 °C with shaking at 110 r.p.m. for 72 h for protein expression. The supernatant was then concentrated using a 10 kDa MW cutoff Centramate cassette (Pall Corporation). The RBD protein was purified by Ni-NTA, followed by size exclusion chromatography, and buffer exchanged into 20 mM Tris-HCl pH 7.4 and 150 mM NaCl. For binding experiments, RBD with AviTag was biotinylated as described previously [32 (link)] and purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris-HCl pH 7.4 and 150 mM NaCl.
7
Recombinant Hemagglutinin Protein Production
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Briefly, the HA ectodomains (HA1 residues 11-329 and HA2 residues 1-176, based on H3 numbering) of A/California/04/2009 (H1N1) and A/Solomon Island/3/2006 (H1N1) were fused with an N-terminal gp67 signal peptide and a C-terminal BirA biotinylation site, thrombin cleavage site, trimerization domain, and His6 tag, and then cloned into a customized baculovirus transfer vector (Ekiert et al., 2011 (link)). Recombinant bacmid DNA was generated using the Bac-to-Bac system (Life Technologies). Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGene HD (Promega). HA was expressed by infecting suspension cultures of High Five cells (Life Technologies) with baculovirus at an MOI of 5 to 10 and incubating at 28°C shaking at 110 rpm for 72 hours. The supernatant was concentrated. HA0 was purified by Ni-NTA, and buffer exchanged into 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. The HA0 was then treated with trypsin (New England Biolabs) to remove the C-terminal tag (BirA biotinylation site, thrombin cleavage site, trimerization domain, and His6 tag) and to produce the cleaved mature HA (HA1/HA2). The trypsin-digested HA was then purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3.
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