Hitrap excel column

Plasmids encoding cDNAs for pre-fusion (SC-TM) or post-fusion RSV subgroup A strain A2, and subgroup B strain 18537 pre-fusion (DsCav1, a gift from Barney Graham) were expanded in E. coli DH5α cells, and plasmids were purified using Qiagen Plasmid Maxiprep kits (Qiagen). Plasmids encoding cDNAs for the protein sequences of mAb 101F, mAb MPE8, and mAb D25, and motavizumab heavy and light chain sequences were cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. Vectors encoding the heavy and light chain sequences of 54G10 were a gift from Dennis Burton. MAb 131-2a protein was obtained from Sigma. Commercial preparations of palivizumab (Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center. For each liter of protein expression, 1.3 mg of plasmid DNA was mixed with 2 mg of polyethylenimine in Opti-MEM I cell culture medium (Fisher). After 10 min, the DNA mixture was added to HEK293F cells (ThermoFisher R79007) at 1 x 10⁶ cells/mL. The culture supernatant was harvested after 5 days, and the protein was purified by HiTrap Excel column (GE Healthcare Life Sciences) for RSV F protein variants or HiTrap MabSelectSure columns for mAbs.

The expression and purification of sENG have been described recently³⁷ . Briefly, residue 1–586 of human ENG cDNA was cloned into pCEP4 with a C-terminal 6xHis tag and sequence was confirmed by DNA sequencing. Plasmids containing sENG were transiently transfected into HEK EBNA cells in DMEM containing 5% FBS before changing into CDCHO expression media. Conditioned medium was harvested every 3–4 days for up to 2 weeks and used for purification. To purify sENG, 5 l of conditioned medium were loaded onto a 5 ml HiTrap Excel column (GE Healthcare) pre-equilibrated in Buffer A (50 mM TrisHCl, pH7.4, 500 mM NaCl, 5 mM imidazole). After wash, sENG was eluted with 5–1000 mM imidazole gradient in buffer A. Fractions containing sENG were identified on SDS-PAGE, pooled and dialysed against 50 mM Tris.HCl, pH7.4, 50 mM NaCl and then loaded onto a 5 ml HiTrap Q HP column. After eluting with a NaCl gradient, fractions containing sENG were identified by non-reducing SDS-PAGE, pooled and further purified on a Superdex 200 16/600 gel filtration column.