Recombinant envelope proteins were expressed in HEK293F or HEK293S cells as described elsewhere (Sanders et al., 2013 (link)). Briefly, the CRF250 SOSIP.664 or BG505 SOSIP.664 encoding plasmids was cotransfected, with a plasmid encoding for Furin (3:1 ratio) specific for R6-cleavage site (RRRRRR), into HEK293F cells using PEI-MAX 4000 transfection reagent (Polysciences, Inc.). To produce the CRF250 SOSIP.664 trimer proteins with varying glycan composition, the glycosidase inhibitors kifunensine and swainsonine were added to the HEK293F cell cultures at a final concentration of 25 μM at the time of transfection (Doores and Burton, 2010 (link)). The CRF250 SOSIP.664 was grown in GnT1-deficient (GnT1−/−) HEK293S cells to produce a Man5–9GlcNAc2 glycan containing trimer. The CRF250 SOSIP.664 gp140 WT and glycovariant trimers were purified from supernatants using a 2G12 Ab antibody affinity column while BG505 trimer and CRF250 WT trimer used for other experiments were purified with a bnAb PGT145 affinity column as described previously (Pugach et al., 2015 ; Sanders et al., 2013 (link)). The affinity-purified proteins were size exclusion chromatography (SEC)-purified with a Superdex 200 10/300 GL column (GE Healthcare) in PBS.
Pei max 4000 transfection reagent
Manufactured by Polysciences
PEI-MAX 4000 is a transfection reagent designed for efficient delivery of DNA, RNA, and other macromolecules into cells. It is a high-molecular-weight polyethylenimine (PEI) polymer that forms complexes with nucleic acids, facilitating their uptake by the target cells.
Automatically generated - may contain errors
2 protocols using pei max 4000 transfection reagent
1
Production and Purification of HIV Envelope Trimers
2
SIV Env SOSIP.664 Trimer Purification
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SOSIP.664 Env trimer modifications were incorporated into the SIVmac239 Env coding sequence, as described previously (32 (link)). Amino acid substitutions were incorporated by using a QuikChange site-directed mutagenesis kit (Agilent Technologies, USA), according to the manufacturer’s instructions. All the mutations were confirmed by DNA sequence analysis (Eton Bioscience, San Diego, CA). Soluble recombinant Env trimers were expressed in HEK293F cells as described elsewhere (32 (link)). Briefly, plasmids encoding the SIVmac239 SOSIP.664 trimer and its V2 variants were cotransfected with a furin expression construct into HEK293F cells at a 3:1 ratio using PEI-MAX 4000 transfection reagent (Polysciences, Inc.). The secreted trimer proteins were purified from cell supernatants after 5 days using agarose-bound Galanthus nivalis lectin (GNL) (Vector Labs) columns as described previously (32 (link)). Affinity-purified proteins were separated by size exclusion chromatography using Superdex 200 10/300 GL columns (GE Healthcare) in phosphate-buffered saline (PBS). The purified trimers were stored at −80°C until use.
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