TGF-beta family technoteMarie Singer2024-02-06T10:10:01+00:00
Technote | TGF beta family
The first quality-matched animal-free TGF–b 1,2 and 3 proteins forimproved cell culture media development
In mammals, distinct genes encode TGF-β 1–3 proteins, which are expressed in unique, occasionally overlapping, patterns and servedifferent functionsin vivo.TGF-β proteins are potent signaling molecules that act primarily through the Smad 2/3-mediated pathway. They are structurally complex, withthe active form of the protein existing as a disulfide-bonded dimer. Recombinant TGF-bproteins are essential reagents in most adult stemcell, iPSC, ESC, and HSCculturemedia. To date,scientists have not been able to compare all three TGF-b proteinsdirectly with guaranteed equivalent purity to define theform that is most efficacious for their specific application.
Quantitative TGF-b-responsive luciferase reporter assay revealsonly modest differences in EC50 between animal-free TGF-b1,2 and 3 in HEK cells. Transfected HEK293T cells were treated in triplicate with a serial dilution of either TGF-β1, TGF-β2 or TGF-β3 for 6 hours. Firefly luciferase activity was measured and normalized to the control Renilla luciferase activity.
Comparable EC50 of 0.84 pM (Qk010 TGF-b1, black, 21.7 pg/ml), 1.69 pM (Qk072 TGF-b2, blue, 42.9 pg/ml) and 1.26 pM (TGF-b3, green, 32.1 pg/ml) were observed.
Using our expertise in this protein family and our certified animal-free manufacturing platform, we have produced all three TGF-b proteins with matched biochemical purity to enable comprehensive media optimization and comparative studies.
The EC50 of TGF-β1, 2, and 3 proteins are broadly comparable in a quantitative SMAD2/3 reporter assay. The relative EC50 values observed, with TGF-β2 exhibiting the highest EC50 (least potent), are consistent with existing literature reports. The effective EC50 and bioactivity of these proteins will depend on the differential expression of TGF-β receptors and co-receptors on the specific cell line of interest.
TGF-β1 PLUS incorporates minor protein engineering to enable its production in an animal-free system, without impacting its biological activity or efficacy in stem cell culture media.
Using animal-free TGF-b proteins removes the uncertainty stemming from co-purification of related proteins, such as other TGF-β isoforms, BMPs, and Activins, which are naturally secreted by mammalian protein expression systems like CHO, HEK, and NSO. This is particularly significant for TGF beta family proteins, which may signal with femtomolar EC50 values.
Quality and purity-matched TGF-b1,2 and 3 provide a unique opportunity to optimizeserum-free and chemically–defined stem cell maintenance and differentiation media.