Mammalian Cell Culture Expression of HEF Protein

Proteins were produced using a mammalian cell culture system previously described [28 (link)]. Briefly, pCD5-HEF expression vectors were transfected into HEK293T cells (ATCC^® CRL-3216™) by incubation with polyethyleneimine (PEI) in a 1:10 DNA/PEI ratio for 16 h at 37 °C. The transfection reagent was then removed and replaced with medium [293 SFM II suspension medium supplemented with 2.0 g/L primatone, 3.6 g/L sodium bicarbonate, 3.0 g/L primatone (Kerry, Naas, Kildare, Ireland), 1% GlutaMax (Gibco, Waltham, MA, USA), 1.5% DMSO, and 2 mM valproic acid] with a further incubation of 5 days at 37 °C after which the supernatant was harvested. The presence of HEF protein in the supernatant was analyzed by Western blot using α-strep-tag mouse antibodies (IBA Life Sciences, Göttingen, Lower Saxony, Germany) at a 1:2000 dilution. Finally, the HEF proteins were purified using sepharose strep-tactin beads (IBA Life Sciences, Göttingen, Lower Saxony, Germany) as previously described [29 (link)]. The two control proteins A/H5 Indo and A/H5 VN Y161A were created and produced as previously described [30 (link)].