Overexpression of human IRAP and aminopeptidase N (APN) was achieved in Freestyle HEK293‐F Cells (Invitrogen R790‐07). The cells were grown in 80 ml of Freestyle Expression Medium (Invitrogen 12338–018) in 250 ml Erlenmeyer flasks (Corning 431144.40). The flasks were kept at 37 °C, 8 % CO2, 70 % relative humidity and with shaking at 130–135 rpm in an Infors Multitron incubator. The cells were transfected with 1.1 μg IRAP overexpressing plasmid/106 cells30 or 0.7 μg APN overexpressing plasmid17 (kindly provided Dr. Alexandros Nikolau at Vrije Universiteit Brussels, Brussels, Belgium)/106 cells in the presence of 2 μg polyethyleneimine/106 cells (Polyethyleneimine, linear, MW‐25 000, Cat. No. 23966, Polysciences). Prior to overexpression, the plasmids were transfected and propagated in Mach1 E.coli (Invitrogen C8620‐03). The plasmid DNA was prepared using QIAGEN Plasmid Plus Maxi Kit (12963). 48 h post‐transfection, the cells were harvested by centrifugation at 130×g for 3 min. The cells were washed once with PBS and the cell pellets were stored at −20 °C until used for membrane preparations.
Freestyle hek 293 f cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
Freestyle HEK 293-F cells are a suspension-adapted variant of the Human Embryonic Kidney 293 (HEK 293) cell line. They are designed for high-density, serum-free growth in suspension culture, enabling rapid and scalable protein production.
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Lab products found in correlation
Freestyle 293 expression medium, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor, by Roche (2 mentions)
L glutamine, by Cytiva (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Plasmid plus maxi kit, by Qiagen (1 mentions)
Freestyle expression medium, by Thermo Fisher Scientific (1 mentions)
Erlenmeyer flask, by Corning (1 mentions)
Agarose beads, by Merck Group (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Stericup gp device, by Merck Group (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Ni nta, by Thermo Fisher Scientific (1 mentions)
Pfusess clig hk, by InvivoGen (1 mentions)
Pfusess chig hg1, by InvivoGen (1 mentions)
Mabselect protein a column, by GE Healthcare (1 mentions)
Freestyle max reagent, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
19 protocols using freestyle hek 293 f cells
1
Overexpression of IRAP and APN in HEK293-F Cells
2
Mov10 Helicase Mutant Generation
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The myc-tagged Mov10 helicase mutant was generated using site-directed mutagenesis to mutate the conserved lysine in motif I to alanine (K531A). The HA-tagged N-terminal half and C-terminal half plasmids were obtained from the source in Ref. [23 (link)]. Constructs were transfected using polyethylenimine (PEI, # 408727, Sigma) in FreeStyle HEK 293 F cells (Invitrogen) and cultured according to the manufacturer’s protocol and as described [2 (link)]. The cells were harvested after 48 h and lysed in lysis buffer (50 mM Tris-Cl 7.5, 150 mM NaCl, 30 mM EDTA, 0.5% Triton) containing protease inhibitors (Roche, Indianapolis, IN, USA) and spun at 14,000 rpm for 5 min at 4 °C. The supernatant was immunoprecipitated with anti-HA magnetic beads (Thermo Fisher Scientific, Carlsbad, CA, USA) for the HA-tagged C-terminal and N-terminal half plasmids of Mov10, and the peptide was eluted with HA peptide (2 mg/mL, Protein Sciences, Roy J Carver Biotech Center, UIUC) for 2 h at 4 °C. The Mov10 helicase mutant was immunoprecipitated using agarose beads coupled to myc antibody (RRID: AB_10109522, Sigma-Aldrich, St. Louis, MO, USA) and the peptide eluted using the myc peptide (2 mg/mL Protein Sciences). Protein concentrations were calculated using a Bradford assay (Bio-Rad Laboratories) and visualized on silver stain.
3
Purification of Myc-tagged Murine MOV10
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Myc-tagged murine MOV10 (27 (link)) construct DNA was transfected using PEI (polyethylenimine, # 408727, Sigma-Aldrich) in Freestyle HEK 293F cells (Invitrogen) and cultured according to the manufacturer's protocol and as described (22 (link)). Cells were harvested after 48 h and lysed in Lysis buffer (50 mM Tris–HCl pH 7.5, 300 mM NaCl, 30 mM EDTA, 0.5% Triton) containing Protease inhibitors (Roche, Indianapolis, IN, USA) and spun at 8000 rpm for 10 min at 4°C. The supernatant was immunoprecipitated overnight with anti-myc agarose (A7470, Sigma-Aldrich). After four washes, myc-MOV10 was eluted with myc peptide 2 mg/ml, (synthesized by the Protein Sciences, Roy J. Carver Biotech Center, UIUC) for 2 h at 4°C and approximated concentration by comparing band using Coomassie Brilliant Blue staining.
4
Transient Spike Protein Expression in FreeStyle HEK293F Cells
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FreeStyle™ HEK293F cells (Invitrogen) were grown in sterile polycarbonate Erlenmeyer flasks on an orbital shaking platform set to 125 rpm. The cells were maintained at a density of 1-3×106 cells/ml at 37°C, with 8% CO2. The cultures were passaged every 3-4 days, at a seeding density of 3×105 cells/ml, using fresh FreeStyle™ 293 Expression Medium (Invitrogen). Spike protein was transiently expressed by transfecting cells, at a density of 1×106 cells/ml, with 1 µg/ml of plasmid DNA. Polyethylenimine was used for transfections at a 3:1 ratio of transfection reagent:DNA. The culture media was harvested 5 days post-transfection and clarified by centrifugation at 2500×g, for 30 minutes. The clarified media was then filtered using a 0.45 µm Stericup-GP device (Merck Millipore). Spike trimers were purified as described for the plant-produced material.
5
Thrombin-Activatable Microplasminogen Fusion Constructs
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The DNA sequence coding for the human thrombin‐activatable microplasminogen (HtPlg) was obtained from GeneArt (ThermoFisher Scientific, Waltham, MA). The HtPlg construct was then fused with two different single‐chain antibodies, the activation‐specific GPIIb/IIIa–targeted (SCE5) and –nontargeted (Mut‐scFv), as previously described.27 , 28 The fusion constructs SCE5‐HtPlg and Mut‐scFv‐HtPlg were transfected in human embryonic kidney cells (freeStyleHEK 293‐Fcells; Life Technologies, Carlsbad, CA), suspension cells for production of the proteins, which were isolated by fast liquid protein chromatography with a nickel‐based metal affinity column Ni‐NTA (Invitrogen, Carlsbad, CA). The detailed procedures are available in the supplementary material.
6
Recombinant Antibody Production in HEK Cells
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VH and VL of the positive phage were subcloned, respectively, into the pFUSEss-CHIg-hG1 and pFUSEss-CLIg-hK (Invivogen). Antibodies were transiently expressed in FreeStyle™ HEK 293-F cells (Life Technologies) using 293fectin transfection reagent according to manufacturer’s instructions. After transfection, cells were grown in the serum-free medium for an additional 5 days. The supernatant was collected and purified on a MabSelect Protein A column (GE healthcare). Eluted IgG was dialyzed against PBS and stored at −80°C.
7
Cell Co-Transfection and Aggregation Assay
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FreeStyle HEK293F cells (Life Technologies Cat# R79007) grown to a density of 1 × 106 cells/mL in a 30 mL volume were co-transfected with 30 μg of either pCMV5-Emerald or pCMV5-tdTomato and 30 μg of the indicated construct using a FreeStyle Max reagent (Life Technologies Cat# 16447100). All cDNAs were encoded in the pCMV5 vector. FreeStyle HEK293F cells were grown at 37°C/8%CO2 with shaking at 125 rpms. Transfected cells were mixed in non-coated 12-well plates at a 1:1 ratio 2-days post-transfection and subsequently incubated for an additional 2 hr. Live cells were imaged in 12-well plates using a Nikon A1 confocal microscope. Aggregation index was calculated using ImageJ, measuring the percentage of signal/frame occupied by cells forming complexes of two or more cells relative to the total signal of the frame.
8
Protein Production and Purification
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Human IgG1-Fc and hEGFR-Fc were cloned into the gWIZ vector (Genlantis, San Diego, CA) with a hexahistidine tag at the C terminus. FreeStyle HEK293F cells (Life Technologies) were cultured in FreeStyle 293 expression medium (Life Technologies) and transiently transfected using polyethylenimine (PEI). After 1 week, the cultures were centrifuged and filtered. 10× PBS was added to the supernatants to achieve a 1× PBS concentration. The resulting solution was applied onto Protein A resin (GenScript, Piscataway, NJ), washed four times with PBS, and finally eluted with 8 ml of 0.1 m glycine, pH 3.5. The proteins were directly eluted into 0.8 ml of 1 m Tris, pH 8.0, to minimize the incubation time at pH 3.5.
Fatty acid-free MSA was purchased from Alpha Diagnostic International (San Antonio, TX). All antigens (hEGFR-Fc, Fc, and MSA) were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Life Technologies), and subsequently monomeric protein was purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 column (GE Healthcare).
Fatty acid-free MSA was purchased from Alpha Diagnostic International (San Antonio, TX). All antigens (hEGFR-Fc, Fc, and MSA) were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Life Technologies), and subsequently monomeric protein was purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 column (GE Healthcare).
9
Purification of HIV-1 Env Trimer Proteins
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The BG505 SOSIP.664 and B41 SOSIP.v4.1 trimers were produced in stable CHO cell lines and purified by 2G12 bNAb affinity chromatography followed by size exclusion chromatography (SEC), as described previously (26 (link), 28 (link)). C-terminally His-tagged versions of the same trimers were used only in ELISAs; they were produced by transient transfection of FreeStyle HEK293F cells (Thermo Fisher) and purified by PGT145 bNAb affinity chromatography followed by SEC (26 (link), 49 (link)). The trimer concentration was adjusted to 0.5 mg/ml in Tris-buffered saline (TBS; 20 mM Tris, 100 mM NaCl, pH 7.5), aliquoted, and frozen.
10
Recombinant human IgG1 antibody production
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Single cell analysis and sequencing of immunoglobulin genes of multiple sclerosis CSF B-cells yielded paired light chain and heavy chain variable regions. Sequences were then combined with published sequences of immunoglobulin heavy constant gamma 1 and the appropriate kappa or lambda light chain constant regions for full recombinant human IgG1 antibody expression. Expression vectors with full heavy and light chains were transfected into Freestyle HEK 293-F cells (Thermo Fisher). Transfected cells were incubated at 37°C and 8% CO2 with constant shaking for 5–7 days for antibody expression. Secreted antibodies were then purified via a protein G agarose column. Purified antibodies were then dialysed into PBS. Detailed methods are described in the Supplementary material .
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