The antigen used in this study was the receptor binding domain (RBD) of native (WuHan-Hu-1) SARS-CoV-2 S-protein (anti-RBD). The antigen was produced based on the previously described methodology, with modifications [21] (link). Briefly, the RBD coding sequence, flanked with the signal peptide coding sequence at 5′-end and 6xHis-Tag at 3′-end, was cloned into pCAGGS expression plasmid. Expi293F cell line (ThermoFisher Scientific), a modified HEK293 line, optimized for production of protein exported outside the cell to the cell culture medium, were transfected with expression vector using ExpiFectamine™ reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. After 5 days, the cell suspension was centrifuged at 4000g, 20 min, 4 °C, and the supernatant was collected. The supernatant containing RBD protein was passed through a chromatography column filled with Ni-NTA resin (Therm Fisher Scientific), to immobilize the protein via His-tag. After immobilization, the column was washed with phosphate-buffered saline (PBS), containing 25 mM imidazole and 0.15 M NaCl. The protein was eluted from the resin using PBS containing 230 mM imidazole and 0.2 M NaCl. Subsequently, the protein solution was concentrated and purified with 10 kDa Amicon centrifugal filter columns (Merck, Germany).
Expifectamine reagent
Manufactured by Thermo Fisher Scientific
Expifectamine reagent is a transfection reagent designed for the efficient delivery of nucleic acids, such as plasmids and messenger RNA (mRNA), into mammalian cells. It facilitates the uptake of these molecules by the cells, enabling their expression or use in various research and application-based studies.
Automatically generated - may contain errors
Lab products found in correlation
Expi293f cell line, by Thermo Fisher Scientific (1 mentions)
Amicon centrifugal filter columns, by Merck Group (1 mentions)
Penta his antibody, by Qiagen (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Dylight antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Superdex 200 10 300 gl column, by Cytiva (1 mentions)
Protein g column, by Thermo Fisher Scientific (1 mentions)
6 protocols using expifectamine reagent
1
Purification of SARS-CoV-2 RBD Antigen
2
Purification of Recombinant TIM-3 Protein
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As previously described74 (link), TIM-3 was expressed and purified in Expi293 cells, according to the Expi293 Expression System manual. Cells were seeded to 3.0 × 106 cells/mL and then were transfected with a mixture of DNA vector (pcDNA-TIM-3), Expifectamine reagent (ThermoFisher Scientific), and Opti-MEM. After 4–6 days, culture was harvested by centrifugation, and culture supernatant was dialyzed against 10 mM HEPES pH 7.6, 0.15 M NaCl (4 times the culture volume). Nickel affinity chromatography, ion exchange chromatography, and size exclusion chromatography were then used to purify the diafiltered supernatant. The protein sample was sterilized by filtration (0.22 µm). A NanoDrop spectrophotometer was used to detect absorbance at 280 nm, and protein concentration was determined using an estimated extinction coefficient (25815 M−1 cm−1).
3
Production and Purification of SARS-CoV-2 RBD and S Proteins
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RBD and S proteins were produced by using mammalian expression plasmids [9 (link),10 (link)] that were transiently transfected into expiCHO™ cells (ThermoFisher, A29133) via manufacturer’s instructions. Briefly, expiCHO cells were transfected with plasmid DNA (1 μg/ml of cell volume) at 6x106 cells/ml in suspension culture using the Expifectamine reagent (ThermoFisher, A14525). Transfected expiCHO cells are then cultured per the manufacturers ‘max titer’ protocol at 32 degrees shaking at 125 rpm for 12 days. Cell culture supernatants were harvested and filtered through a 0.2 μM membrane and both S protein and RBD were purified using a 20 mL Ni2+-charged HiPrep IMAC FF 16/10 column (Cytiva) to bind the His-tagged region engineered into each protein [9 (link),10 (link)]. A 10 kDa MWCO centrifugal filter unit (Amicon, ACS501024) was used to concentrate fractions containing RBD. Protein purity was validated by SDS-PAGE and western blotting using a PENTA-His antibody (Qiagen, ID:34660). Purified RBD and S proteins were characterized by sedimentation velocity analytical ultracentrifugation using a Beckman Coulter XL-I. Data were analyzed using SEDFIT’s continuous c(s) distribution model [13 (link)], SEDANAL version 7.45 [14 (link)], or DCDT+ version 2.4.3 [15 (link)]. Purified protein was stored at -20°C in 50% glycerol with 5 mM sodium azide.
4
Producing and Labeling SARS-CoV-2 Antibodies
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Monoclonal antibody CR3022 was produced by transient transfection of 293 F cells with cDNA expression plasmids obtained from BEI resources. The cells were transfected at a density of approximately 2 × 106 cells/ml with equal amounts of each plasmid using Expifectamine reagent (Thermo Fisher) according to the instructions provided by the manufacturer. The supernatant was harvested after 4–5 days and antibody purified on Protein G Sepharose (Abcam, GE Healthcare) following standard protocols. Monoclonal antibodies I1 and I14 against the VSV glycoprotein and monoclonal antibody 23H12 against the VSV matrix protein were purified from hybridoma supernatant by affinity chromatography on Protein G sepharose. The antibodies were labeled with fluorophores using Dylight antibody labeling kits from Thermo Fisher. The following SARS-CoV-2-specific human monoclonal antibodies were kindly provided by Distributed Bio: DB_A03-09, 12; DB_B01-04, B07-10, 12; DB_C01-05, 07, 09, 10; DB_D01, 02; DB_E01-04, 06, 07; DB_F02-03.
5
SARS-CoV-2 Spike Protein Expression and Purification
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Recombinant spike and spike RBD proteins of SARS-CoV-2 for antigen-specific B cell flow cytometry and ELISPOT were expressed and purified as previously described (15 (link)). Briefly, spike glycoprotein trimer (uncleaved spike stabilized in the prefusion conformation (GGGG substitution at furin cleavage site and 2P mutation; ref. 65 (link)) and RBD protein (12 (link)) were cloned into a pHLsec vector containing Avi and 6xHis tags. Biotinylated spike and RBD were expressed in Expi293F cells (Thermo Fisher Scientific). Supernatants were harvested after 7 days and purified. For the production of biotinylated protein, spike- and RBD-encoding plasmids were cotransfected with BirA and PEI-Max in the presence of 200 μM biotin.
Recombinant S1 protein constructs spanning SARS-CoV-2 residues 1–530 for ELISPOT were produced as previously described (28 (link), 30 (link)). Briefly, codon-optimized DNA fragments were cloned into mammalian expression vector pQ-3C-2xStrep to create plasmids, which were then transfected into Expi293F cells growing at 37°C in a 5% CO2 atmosphere using ExpiFectamine reagent (Thermo Fisher Scientific). Proteins were purified by strep-tag affinity followed by size exclusion chromatography.
Recombinant S1 protein constructs spanning SARS-CoV-2 residues 1–530 for ELISPOT were produced as previously described (28 (link), 30 (link)). Briefly, codon-optimized DNA fragments were cloned into mammalian expression vector pQ-3C-2xStrep to create plasmids, which were then transfected into Expi293F cells growing at 37°C in a 5% CO2 atmosphere using ExpiFectamine reagent (Thermo Fisher Scientific). Proteins were purified by strep-tag affinity followed by size exclusion chromatography.
6
Recombinant Monoclonal Antibody Production
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Recombinant monoclonal antibodies were prepared as described previously (Adachi et al., 2019 (link); Tiller et al., 2008 (link)). In brief, VH/VL genes of selected B cell samples from single-cell cultures or published monoclonal antibodies were cloned into human IgG1 or IgG3 heavy chain, and kappa or lambda light chain expression vectors. Expression plasmids encoding the heavy and light chains of the Fab were also prepared. Pairs of heavy and light chain expression vectors were transfected into Expi293F cells using ExpiFectamine reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 4 days post-transfection. The IgG and Fab antibodies were purified from the culture supernatant using a protein G column (Thermo Fisher Scientific) and a Talon resin affinity chromatography (Clontech), respectively, and then subjected to further analysis after dialysis against PBS. To remove soluble aggregates for further experiments, size exclusion chromatography was performed. After the centrifugation, the supernatant of either IgG1 or IgG3 was loaded onto a Superdex 200 10/300 GL column (Cytiva) at a flow rate of 0.5 mL/min using PBS as running buffer. Gel Filtration Standard (BioRad) was used according to the manufacturer’s instruction.
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