Recombinant Human GDF-15, Animal-Free Protein

GDF15 signals through GRAL and co-receptor RET leading to RET phosphorylation and signalling through the ERK and AKT pathway (reviewed in Emmerson et al., 2018). Commercial sources of GDF15, in particular those purified from mammalian expression systems, have been shown previously to be contaminated with trace amounts of TGF beta. These trace contaminants cause misleading experimental results due to the picomolar or even femtomolar EC50s (Olsen et al., 2017). Here we use a well-characterized SMAD2/3 activation assay to show that there is no contamination from other TGF beta family proteins.Bioactivity is determined using a luciferase reporter assay in HEK293T cells. Cells are treated (in triplicate) with a serial dilution of GDF15 or Qk010 TGF beta 1 for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. There is no response with high concentrations of GDF-15, indicating no contamination with other TGF beta family proteins.

GDF15 migrates as a single band at 24 kDa in non-reducing (NR) and 13 kDa as a single monomeric species upon reduction (R). No contaminating protein bands are visible.Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+ beta -mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.