The paired antibody VH/VL chains were cloned into Igγ, Igα1 or Igα2 and Ig expression vectors using T4 ligase (NEB). For production of IgG and monomeric IgA, the plasmids with paired heavy chain (IgG, IgA1, IgA2) and light chain genes were co-transfected into Expi293TM expression system (Thermo Fisher Scientific) following the manufacturer’s protocol to produce recombinant monoclonal antibodies. For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293TM expression system (Thermo Fisher Scientific) at the ratio of 1:1:1 following the manufacturer’s instructions. Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelectTM IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 ml in PBS (Life Technologies) (Table S2), and then stored at 4 °C or −80 °C for further characterization.
Expi293 expression system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Expi293 Expression System is a transient mammalian protein expression system designed for high-yield production of recombinant proteins in Expi293F cells. The system utilizes a proprietary expression vector and optimized transfection reagents to enable efficient protein expression.
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Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Histrap excel column, by GE Healthcare (5 mentions)
Protein a sepharose, by GE Healthcare (4 mentions)
Hitrap protein a hp column, by GE Healthcare (4 mentions)
Kta start chromatography system, by GE Healthcare (4 mentions)
Protein a affinity chromatography, by GE Healthcare (4 mentions)
Ni nta agarose, by Qiagen (4 mentions)
Amicon ultracell, by Merck Group (4 mentions)
Histrap ff column, by GE Healthcare (4 mentions)
Expi293f cells, by Thermo Fisher Scientific (3 mentions)
Hitrap protein a hp column, by Cytiva (3 mentions)
Superdex 200 10 300 column, by GE Healthcare (3 mentions)
Ni sepharose histrap ff column, by GE Healthcare (3 mentions)
Pcdna3.1 expression plasmid, by Thermo Fisher Scientific (3 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (3 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (3 mentions)
Talon metal affinity resin, by Takara Bio (3 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (3 mentions)
Expi293f, by Thermo Fisher Scientific (3 mentions)
Irdye 800cw, by LI COR (2 mentions)
160 protocols using expi293 expression system
1
Recombinant Monoclonal Antibody Production
2
Recombinant Plasmablast Antibody Production
3
Fc-Fusion Antibody Constructs
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IgG1 Fc (pEXPR-IgG1 Fc) was fused to either SpyTag (AHIVMVDAYKPTK) or KTag (ATHIKFSKRD) with additional GY-dipeptide at the C-terminus by using standard molecular cloning techniques. Native IgG1 glycosylation at position N297 was eliminated by introducing a N297A mutation by Quick Change Side-Directed Mutagenesis. Plasmids coding for chimeric cetuximab (C225, Erbitux®) and trastuzumab (Herceptin®) were kindly provided by Merck KGaA (Darmstadt). Cetuximab and trastuzumab variants containing SpyTag at the C-terminus of either the heavy chains, the light chains, or at both chains were prepared by standard molecular cloning techniques. A (GSG)2-linker was introduced between the C-terminus of the antibody and SpyTag. IgG1 Fc and full-length antibodies were transiently expressed from HEK293F cells using the Expi293 Expression System (Life Technologies). Supernatants containing secreted proteins were conditioned and applied to spin columns with PROSEP-A Media (Montage, Merck Millipore). Columns were washed with 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0 and proteins eluted with 0.2 M Glycine/HCl pH 2.5 into 1 M Tris/HCl pH 9.0. Eluted proteins were dialyzed in 1 × DPBS (Life Technologies) using Amicon Ultra-15 (Merck Millipore, NMWL 10000 Da) and stored at 4 °C.
4
Expi293F Expression and gp120 Detection
5
Expi293 Expression System for Protein Purification
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The Expi293 expression system (Life Technologies, A14524) was used to generate the conditioned media for purification, according to the manufacturer’s instructions. In brief, Expi293F cells (Thermo Fisher) were suspension cultured in Expi293 expression medium at 37 °C in a humidified atmosphere with 8% CO2. When the cell density reached 2.5 million/mL, plasmid DNA and ExpiFectamine 293 reagent were mixed, incubated for 5 min, and added to the cells. The final concentration of the DNA and transfected reagent was 1 µg and 2.7 µL per milliliter, respectively. Five hours after transfection, 100 µg/mL porcine heparin (Sigma, H3149) and protease inhibitor mixture, 1:400 (Sigma, P1860), were added to the cells. Sixteen hours after transfection, enhancer reagents 1 and 2 were added. Ninety-six hours after transfection, conditioned media were harvested. Aliquots were tested for Fc fusion protein concentrations using a human Fc ELISA Kit (Syd Labs, EK000095-HUFC-2) according to the manufacturer’s instructions. Protease inhibitors were added (1:500) to the bulk, which was stored at −80 °C until further use.
6
Recombinant Expression of Wnt Proteins
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Expression constructs for mouse and human Wnt proteins with various N-terminal tags were designed to include tobacco etch virus (TEV) protease cleavage sequence after the tag as shown in Supplementary file 1 , and constructed by extension PCR. Expression plasmids for various albumin family proteins with N-terminal tag (either Tg or PA tag) were also constructed by the same strategy. The coding region for Tg-Wnt3a was subcloned into a mammalian episomal expression vector pEBMulti-Hyg (Wako Pure Chemical, Japan) and used to transfect HEK293S GnT1- cells. Transfected cells were selected against medium containing 0.2 mg/ml hygromycin B, and the resistant polyclonal cell population confirmed to maintain high Wnt3a-producing ability over the repeated passages were used for the protein production. HEK293S GnT1- cells stably producing Tg-Wnt5a were established in a similar manner. For the co-expression of Wnt and AFM, expression plasmids for Tg-hAFM and N-terminally PA tagged mouse or human Wnts were co-transfected at a DNA ratio of 10:1 using the Expi293 Expression System (Life Technologies Inc. Tokyo Japan), according to the procedures recommended by the manufacturer. The culture medium was harvested at 90 hr post-transfection, and immediately used for the Wnt/β-catenin reporter assay or the protein purification.
7
Generation of Recombinant Human LIF
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For construction of human LIF expression plasmid, a nucleic acid fragment encoding the full‐length human LIF with a poly His‐tag at the C terminus was synthesized and inserted in pcDNA3.1(+) vector at NotI/XbaI site by GenScript USA, Inc. The Expi293 expression system (Life Technologies, A14524) was used to generate the conditioned media for purification. Pyrogen‐free reagents were employed. To inactivate endotoxin, columns and equipment (Akta Explorer System, GE Healthcare) were sanitized prior to use by exposure to 0.5 N NaOH for approximately 40 min, as described (Xin et al, 2021 (link)). LPS levels were measured by ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Cat. No. L00350).
Conditioned medium was adjusted to 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, and 0.01% Tween 20 before applying to HisTrap™ HP 1 ml column (GE Healthcare). The column was then washed with buffer containing 50 mM imidazole, and the elution of protein was achieved in 100 mM imidazole. Eluted protein was buffer exchanged into PBS containing 0.01% Tween 20. Recombinant protein was more than 95% pure, with LPS levels of approximately 0.02 EU/mg protein.
Conditioned medium was adjusted to 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, and 0.01% Tween 20 before applying to HisTrap™ HP 1 ml column (GE Healthcare). The column was then washed with buffer containing 50 mM imidazole, and the elution of protein was achieved in 100 mM imidazole. Eluted protein was buffer exchanged into PBS containing 0.01% Tween 20. Recombinant protein was more than 95% pure, with LPS levels of approximately 0.02 EU/mg protein.
8
Affinity-based Expression and Purification of FcRn
9
Anti-PSMA scFv Antibody Production
10
Recombinant Influenza Antigen Production
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