Opti mem 1 glutamax cell culture medium

Plasmids encoding cDNAs for RSV subgroup A strain A2 F protein (wild-type post-fusion lacking the signal peptide and transmembrane domain), the SC-TM construct and the subgroup B strain 18537 protein construct (wild-type post-fusion F protein lacking the signal peptide and transmembrane domain) were synthesized (Genscript). The RSV B 18537 Ds-Cav1 (pre-fusion) construct was a gift from B. Graham (NIH). Plasmids were expanded in E. coli DH5α cells and DNA was purified using Qiagen Plasmid Maxiprep kits (Qiagen). For each litre of transfected culture, 1.3 mg of plasmid DNA was mixed with 2 mg of polyethylenimine in Opti-MEM I + GlutaMAX cell culture medium (Fisher). After 10 min, the DNA mixture was added to HEK293 cells at 1 × 10⁶ cells per ml. The culture supernatant was collected after 6 days and the protein was purified by a HiTrap Talon crude (GE Healthcare Life Sciences) column for RSV F protein variants and mutants. Expression and purification of mAbs 101F, motavizumab and D25 have been described previously^{14 (link)}. Commercial preparations of palivizumab (Synagis; Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center.