Antibody binding to SARS-CoV-2 antigen was measured as previously described [27] (link) with modification of antigen display cells from HEK293T to Expi293F (Thermo). The pME18S plasmid expressing Spike protein C-terminal retention signal deletion from Wuhan (WT), Delta, and Omicron variant, Flag-NTD-PILR-TM, Flag-RBD-PILR-TM, and Flag-NTD (W64A, H66A, V213A, and R214A)-PILR-TM, were co-transfected with pMx plasmid expressing GFP as the marker to the Expi293F cells (Thermo). The transfectant cells were incubated with supernatant containing expressed antibodies for 30 minutes then followed by incubation with APC-anti-human IgG (H+L) antibody (Jackson ImmunoResearch, USA). Bound antibodies to the GFP-positive cells were then analyzed by flow cytometry (Attune NxT, Thermo).
Expi293f
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Expi293F expression system is a suspension-adapted HEK293 cell line designed for the transient expression of recombinant proteins. It provides a scalable and efficient platform for the production of a wide range of proteins, including antibodies, enzymes, and other biotherapeutics.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 f, by Thermo Fisher Scientific (13 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (12 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (10 mentions)
Hek293t, by American Type Culture Collection (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Hek293f, by Thermo Fisher Scientific (6 mentions)
Expicho s, by Thermo Fisher Scientific (6 mentions)
Hek293t, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Mabselect sure resin, by GE Healthcare (5 mentions)
Hiprep 26 10 desalting column, by GE Healthcare (5 mentions)
Mycoalert mycoplasma detection kit, by Lonza (5 mentions)
Hek293t cell, by American Type Culture Collection (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Veroe6 tmprss2, by Thermo Fisher Scientific (3 mentions)
Sad s35, by ACROBiosystems (3 mentions)
Hb 112, by American Type Culture Collection (3 mentions)
Rprotein a sepharose, by GE Healthcare (3 mentions)
Pcmv3 flag tmprss2, by Sino Biological (3 mentions)
Hmyd88 expressing 293 cells, by InvivoGen (3 mentions)
127 protocols using expi293f
1
Antibody Binding to SARS-CoV-2 Variants
2
Cell Line Maintenance and Validation Protocol
3
Antibody Phage Display Cloning
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Unique scFv sequences isolated by antibody-phage display in MTPs or the complete output of the third panning rounds (all scFv encoding DNA fragments) were subcloned into pCSE2.7-hIgG1-Fc-XP using NcoI/NotI (New England Biolabs, Frankfurt, Germany) for mammalian production as scFv-Fc, an IgG-like antibody format. The vector pCSE2.7-hIgG1-Fc-XP is derived from the vector pCSE2.6-hIG1-Fc-XP61 (link) with an additional AscI restriction site in the cloning site for improved NcoI/NotI cloning. The best neutralizing antibodies were converted into the human IgG1 format by subcloning of VH in the vector pCSEH1c (heavy chain) and VL in the vector pCSL3l/pCSL3k (light chain lambda/kappa)37 (link). EXPI293F (Thermo Fisher Scientific) cells were transfected with both vectors in parallel. For production, the transfected EXPI293F cells were cultured in chemically defined medium F17 (Thermo Fisher Scientific) supplemented with 0.1% pluronic F68 (PAN-Biotech, Aidenbach, Germany) and 7.5 mM L-glutamine (Merck). A subsequent protein A purification was performed as described previously61 (link).
4
Antibody Expression and Purification
5
Recombinant Expression and Purification of hChi3l1 and mChi3l1
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The hChi3l1 (UniProt, #P36222) and mChi3l1 (UniProt, #Q61362) were expressed in Expi293F (Thermo Fisher Scientific) cells with Avitag (GLNDIFEAQKIEWHE) and His tag at the C-terminal of the sequences. The proteins were purified using the Ni-NTA Agarose (Invitrogen) according to the manufacturer’s instructions. The biotinylation of Chi3l1 was performed using BirA ligase (Avidity, #BirA500). The hChi3l1 without tags for X-ray crystallography was expressed in Expi293F (Thermo Fisher Scientific) cells and was in-house purified by affinity chromatography using the HiPrep Heparin FF (Cytiva) and by size exclusion chromatography using the HiLoad 16/600 Superdex 200 pg column (Cytiva) according to the manufacturer’s instructions.
6
IgG Production by Transient Transfection
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For IgG production, selected antibodies were converted into the human IgG1 format by subcloning of VH in the vector pCSEH1c (heavy chain) and VL in the vector pCSL3l/pCSL3k (light chain lambda/kappa)76 (link), adapted for Golden Gate Assembly procedure with Esp3I restriction enzyme (New England Biolabs). Expi293F (Thermo Fisher Scientific) cells were transfected with 12.5 µg of both vectors in parallel in a 1:1 ratio. For production, the transfected Expi293F cells were cultured in chemically defined medium F17 (Thermo Fisher Scientific) supplemented with 0.1% pluronic F68 (PAN-Biotech) and 7.5 mM L-glutamine (Merck) for 7 days. A subsequent protein A purification was performed as described above.
7
Mammalian Expression and Purification of Monoclonal Antibodies
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For each antibody, variable genes were inserted into plasmids encoding the constant region for the heavy chain (pFUSEss-CHIg-hG1, Invivogen) and light chain (pFUSE2ss-CLIg-hl2, Invivogen and pFUSE2ss-CLIg-hk, Invivogen) and synthesized from GenScript. mAbs were expressed in FreeStyle 293F or Expi293F mammalian cells (ThermoFisher) by co-transfecting heavy chain and light chain expressing plasmids using polyethylenimine (PEI) transfection reagent and cultured for 5-7 days. FreeStyle 293F (ThermoFisher) and Expi293F (ThermoFisher) cells were maintained in FreeStyle 293F medium or FreeStyle F17 expression medium supplemented with 1% of 10% pluronic F-68 and 20% of 200 mM L-Glutamine. These cells were cultured at 37°C with 8% CO2 saturation and shaking. After transfection and 5-7 days of culture, cell cultures were centrifuged at 6000 rpm for 20 minutes. Supernatant was 0.45 μm filtered with PES membrane Nalgene Rapid Flow Disposable Filter Units. Filtered supernatant was run over a column containing Protein A agarose resin that had been equilibrated with PBS. The column was washed with PBS, and then antibodies were eluted with 100 mM Glycine HCl at pH 2.7 directly into a 1:10 volume of 1 M Tris-HCl pH 8. Eluted antibodies were buffer exchanged into PBS three times using 10 kDa or 30 kDa Amicon Ultra centrifugal filter units.
8
Purification of Full-length Human TPH2
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The full-length human TPH2 gene was purchased from Sino Biological Co., Ltd, and reconstructed to pCAG with an N-terminal Twin-Strep-tag and a 3×Flag-tag. The construct was then transfected into Expi293F (Thermo Scientific) with PEI reagent and cultured for 72 h at 37°C under 8% CO2. After that, cells were harvested, resuspended, and lysed by sonication in buffer A (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM FeSO4, 0.1 mM tryptophan, 0.1 mM EDTA, 10% v/v glycerol, 2% Tween-20, and 1 mM PMSF). Insoluble material was removed by centrifugation at 15,000 g and the supernatant was loaded on a 2 ml Strep-Tactin®XT column equilibrated with lysis buffer. The column was washed successively with 2 mM ATP in buffer A to remove the endogenously expressed HSP70 protein before TPH2 was eluted in steps with three times of buffer A containing 5, 25, and 50 mM biotin. Fractions containing pure TPH2 protein were identified using SDS-PAGE and further purified using size exclusion chromatography. TPH2 was loaded on a Superose™ 6 Increase 10/300 GL column attached to an AKTA pure system (Cytiva) equilibrated in buffer B (50 mM HEPES pH 7.5, 150 mM NaCl, 0.02% w/v glyco-diosgenin). Fractions were assessed using SDS-PAGE and concentrated for cryo-EM analysis. Approximately 0.25 mg of full-length TPH2 can be obtained from 500 ml of cells.
9
Cell Lines for HER2 Expression
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The human breast adenocarcinoma cell lines SK-BR-3 (ATCC, HTB-30) and BT-474, which naturally express the HER2 antigen, were grown in Dulbecco's Modified Eagle's Medium (DMEM GlutaMAX™, Thermo Fischer Scientific, USA) supplemented with 10% Fetal Calf Serum (FCS). The HER2-expressing human breast ductal carcinoma cell line ZR-75-30 (ATCC® CRL-1504™), the rat basophilic leukemia mast cell line RBL-SX38 (Wiegand et al., 1996 (link)) expressing the human form of the tetrameric (αβγ2) FcεRI receptor, and the lymphoblastoid B cell line RPMI 8866, which highly expresses CD23, were grown in Roswell Park Memorial Institute Medium (RPMI GlutaMAX™, Thermo Fischer Scientific) supplemented with 10% FCS. All cells were maintained at 37°C in 5% CO2. Human embryonic kidney (HEK) cell-derived Expi293F™ cells (Thermo Fischer Scientific) were grown on a Stuart orbital shaker (model SSL1), at 125 rpm in 8% CO2 in serum-free Expi293 expression medium (Thermo Fischer Scientific).
10
Protein Production in Expi Cell Lines and Huh7.5.1-ACE2-TMPRSS2 Cell Culture
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ExpiCHO-S™ (ThermoFisher Scientific, Catalog number: A29127) and Expi293F™ (ThermoFisher Scientific, Catalog number: A14527) cells were used to produce recombinant proteins. Both cell lines were maintained according to the manufacturer recommended protocol until transfection. ExpiCHO-S™ (1ml, 1x107cells) were directly thawed into 25ml of prewarmed ExpiCHO™ expression media and allowed to revive for 2-3 days. The cells were passaged every 3-4 days to maintain density at or below 4x106 - 6x106 viable cells/ml. ExpiCHO-S™ cells were maintained at 37°C, 8% CO2 and 125 +/- 5rpm. Expi293F™ (1ml, 1x107cells) were directly thawed into 25ml of prewarmed Expi293™ expression media and allowed to revive for 2-3 days. The cells were passaged every 3-4 days to maintain density at or below 3x106 - 5x106 viable cells/ml. Expi293F™ cells were maintained at 37°C, 8% CO2 and 125 +/- 5rpm
Huh7.5.1-ACE2-TMPRSS2 cell line was a gift from Andreas Puschnik. 1x107- 1.5x107 cells were thawed directly into prewarmed complete culture media (Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, 10mM HEPES, 1 × Pen-Strep-Glutamine). The cells were allowed to revive for 2-3 days and were passaged once they reached >70-80% confluence and were maintained at >95% viability in complete culture media. Huh7.5.1-ACE2-TMPRSS2 cells were maintained at 37°C and 5% CO2.
Huh7.5.1-ACE2-TMPRSS2 cell line was a gift from Andreas Puschnik. 1x107- 1.5x107 cells were thawed directly into prewarmed complete culture media (Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, 10mM HEPES, 1 × Pen-Strep-Glutamine). The cells were allowed to revive for 2-3 days and were passaged once they reached >70-80% confluence and were maintained at >95% viability in complete culture media. Huh7.5.1-ACE2-TMPRSS2 cells were maintained at 37°C and 5% CO2.
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