Fig. 2

Efficient production of mature TGF-β member Activin A using FlexiBAC. a Sf9 insect cells were infected with recombinant baculovirus generated using the commercial Bac-to-Bac system (ThermoFisher Scientific) or the FlexiBAC system. Cell supernatants were collected at the indicated times after infection, resolved on 4–20% gradient SDS-PAGE gels under reducing conditions, then analyzed by western blot with anti-Activin A IgG. Bands corresponding to pro-Activin A (uncleaved) and mature Activin A (cleaved) are indicated. b Activin A secretion from insect cells infected with baculovirus generated using the precursor backbone for the FlexiBAC system (called “DefBac-H092”, which expresses viral chitinase and cathepsin) or the current backbone (called “DefBac”, which does not express viral chitinase and cathepsin). c Activin A maturation and secretion from insect cells infected with DefBac-derived baculovirus vs. DefBac^Fur+-derived baculovirus. In addition to expressing the target protein of interest, DefBac^Fur+ expresses furin convertase, which converts pro-Activin A to its mature form. d Insect cells were either singly infected or co-infected with the following virus: pFastBac::Activin A (lane 1), DefBac-H092::Activin A (lane 2), DefBac::Activin A (lane 3), DefBac^Fur+::Activin A (lane 4), DefBac::Activin A and DefBac::polh::Furin (lane 5), DefBac::Activin A and DefBac::p6.9::Furin (lane6). Each virus was added to insect cells at MOI = 0.2 except for DefBac::p6.9::Furin (MOI = 2). The conditioned media was analyzed by western blot. Samples taken from peak Activin A expression times are shown (96 hpi for conditions 1–5, 72 hpi for condition 6). e Multi-step purification of mature Activin A from conditioned media from insect cells infected with DefBac^Fur+::Activin A. Samples were resolved on an SDS-PAGE gel and stained with coomassie blue. Samples 1–5 were reduced with DTT prior to loading. f To assess activity, purified mature Activin A was added to epiblast derived stem cells (EpiSCs) cultured on fibronectin. Fold changes in gene expression, normalized to beta-actin, were determined over 11 days by quantitative polymerase chain reaction (qPCR) for the pluripotency makers Oct4, Nanog, Fgf5, and for the lineage marker, Pax6 (mean +/− S.D.; n = 3 separate experiments). Activin A from a commercial source was used as a control (grey box)