IgG1 Fcs were expressed from a pFUSE-hIgG1-Fc vector (encoding residues 221-447 of IgG1; plasmid purchased from InvivoGen). Wild-type IgG1 Fc was expressed exactly as encoded within this plasmid; mutations for the E382A/S/R constructs were introduced by site-directed mutagenesis (QuikChange II kit; Agilent), using mutagenic primers (Supplementary Table 4 ) synthesised by Eurofins Genomics. Sequences of resulting IgG1 Fc constructs are shown in Supplementary Fig. 11 . Fcs were transiently expressed in FreeStyle293F cells (ThermoFisher), using FreeStyle™ MAX Reagent (ThermoFisher), as described in the manufacturer’s protocol. Cells were left to incubate at 37 °C, 8% CO2, shaking at 125 rpm (New Brunswick S41i incubator), and harvested after seven days by centrifugation at 3100 x g for 30 minutes. Supernatants were filtered through a 0.2 μm membrane and antibodies purified by affinity purification with a HiTrap Protein A HP column (Cytiva), followed by size exclusion chromatography with a Superdex 200 16/600 column (Cytiva) in 10 mM HEPES, 150 mM NaCl (pH 8.0).
Pfuse higg1 fc vector
Manufactured by InvivoGen
The PFUSE-hIgG1-Fc vector is a plasmid designed for the expression and purification of human IgG1 Fc fusion proteins in mammalian cells. The vector contains the human IgG1 Fc region, which can be used to produce Fc-fusion proteins.
Automatically generated - may contain errors
Lab products found in correlation
Spike rbd 5 15 25aa lgbit 12xhistag, by InvivoGen (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Hitrap protein a hp column, by Cytiva (1 mentions)
Superdex 200 16 600 column, by Cytiva (1 mentions)
Freestyle max reagent, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 kit, by Agilent Technologies (1 mentions)
3 protocols using pfuse higg1 fc vector
1
Expression and Purification of Mutant IgG1 Fc
2
SARS-CoV-2 Protein Expression Constructs
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Plasmids were constructed by standard molecular biology methods. The DNA fragments of Spike-RBD, N protein, ACE2, and LgBiT were synthesized by IDT Technologies. The SmBiT tag was generated by overlap-extension PCR. The Spike-RBD-5/15/25aa-LgBiT-12xHisTag, Spike-RBD-15/25aa-SmBiT-12xHisTag, N protein(44–180)-10aa-LgBiT-12xHisTag, N protein(44–180)-10aa-SmBiT-12xHisTag, LgBiT-10aa-N protein(44–257)-12xHisTag, and SmBiT-10aa-N protein(44–257)-12xHisTag were generated by subcloning into a pFUSE-12xHisTag vector (adapted from the pFUSE-hIgG1-Fc vector from InvivoGen). The ACE2-Fc fusion plasmids were generated by subcloning the gene fragments of ACE2 and mutant into the pFUSE-hIgG1-Fc vector. The C004, C105, and C135 IgGs LC and HC plasmids were a generous gift from the Nussenzweig lab (Rockefeller University). The CR3022 IgG plasmids were a generous gift from the Kim lab (Stanford) and the Wilson lab (Scripps). The C135 Fab was cloned by removing the Fc domain from the HC plasmid. Complete plasmid sequences are available upon request.
3
SARS-CoV-2 Protein Expression Constructs
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