Qk017 Recombinant human GDF15 protein is a 25 kDa disulphide-linked dimer composed of the mature domain of human growth differentiation factor 15 protein. Our recombinant GDF15 protein is expressed in E. coli and extensively validated to ensure no trace contamination of TGFβ, which can cause misleading results.
Qk017 Recombinant human GDF15 protein
Growth differentiation factor 15 (GDF-15) is a distant member of the TGFβ superfamily. Its expression is tightly regulated and circulating GDF15 protein in serum is associated with diseases such as cancer, cardiovascular disease, obesity and metabolic disease. GDF15 protein is being recognized an important biomarker for cellular stress.
Unlike other members of the TGFβ superfamily that cause activation of the SMAD pathway, GDF15 protein 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 recombinant human GDF15 protein, in particular those purified from mammalian expression are frequently contaminated with trace amounts of TGFβ and related proteins. These trace contaminants cause misleading experimental results due to the picomolar or even femtomolar EC50s of this family of cytokines (2). Please be cautious with your source of recombinant GDF15 protein, our scientists are happy to provide further information, please email firstname.lastname@example.org
We produce our proteins in E. coli with no animal products in our culture or purification processes to ensure there is no contamination from related proteins. In addition, we use a well-characterized SMAD2/3 activation assay to confirm there is no SMAD signalling.
Summary: Qk015 Mature domain of human GDF15 protein (Uniprot: Q99988) expressed in E. coli and purified to homogeneity. Mature active protein is a disulfide-linked dimer.
Molecular mass: ~25 kDa (for the dimer)
Form: protein is provided lyophilized from a fully volatile solution without carrier protein.
Growth/differentiation factor 15, Macrophage inhibitory cytokine 1 (MIC 1), NSAID activated gene 1 protein (NAG1)
1. Emmerson, P. J., Duffin, K. L., Chintharlapalli, S. & Wu, X. GDF15 and Growth Control. Front. Physiol. 9, 1712 (2018).
2. Olsen, O. E., Skjærvik, A., Størdal, B. F., Sundan, A. & Holien, T. TGF-β contamination of purified recombinant GDF15. PLoS One 12, e0187349 (2017).
Purity and bioactivity
For peace of mind that our recombinant human GDF15 protein will work exactly the same way every day, from batch to batch and at any scale you need, we conduct extensive purity and bioactivity analysis. This includes SDS-PAGE, mass spectrometry, reverse phase chromatography, UV spectroscopy and endotoxin level testing.
To confirm the biochemical identity of our recombinant human GDF15 protein and ensure that its purity meets are rigorous standards, we conduct SDS-PAGE on every protein batch. As GDF-15 is a disulfide-linked dimer, we use both reduced and non-reduced conditions, as well as appropriate high sensitivity stains, for our SDS-PAGE analysis.
In the past, recombinant GDF15 protein from commercial suppliers was contaminated with endogeneous TGF beta family growth factors. Our production methods and careful elimination of any potential sources of contamination ensure a highly pure protein. We confirm this in a sensitive firefly luciferase reporter assay in HEK293T cells.
Find out more about our extensive purity testing in the next tab.
Protein purity: SDS-PAGE in reduced and non-reduced conditions
Bioactivity: we use a well characterized SMAD2/3 activation assay to show that there is no contamination from other TGFβ family proteins.
Our products are for research use only and not for diagnostic or therapeutic use. Products are not for resale.
For final purity and activity tests on our proteins, we choose a vial at random and reconstitute as recommended. Biochemical identity and purity is determined using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. Bioactivity is quantified using an appropriate cell-based assay. As stem cells are sensitive to endotoxin levels, we use a high resolution test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). We also check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so you can rely on your calculated dilution.
Result: 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 (+β-mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.
Bioactivity: luciferase reporter assay
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 amount of TGFβ. 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 TGFβ family proteins.
Result: EC50 = 0 pM (no contamination with TGFβ or related growth factors)
Bioactivity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. Cells are treated (in triplicate) with a serial dilution of GDF15 or Qk001 ActA for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity.
Purity: mass spec analysis
Result: calculated molecular mass of the GDF15 dimer is 24556.4 Da. Result of the analysis: 24556.8 Da which is consistent with the calculated mass. No significant heterogeneity is present.
MALDI mass spectrometric analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident in SDS-PAGE analysis. The results are compared with calculated mass of the protein with the assumption that all the cysteines are disulphide-linked. The different peaks represent different charge states of the protein.
Purity: analytical reverse phase chromatography
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Protein purity and structural homogeneity is analyzed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analyzed in ACE C4 4.6 x 250 mm column using eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid. Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient.
Purity: endotoxin level determination
Result: Endotoxin level <0.005 EU/ug protein (below level of detection)
Stem cell cultures are sensitive to endotoxins1, which can be present in media, serum and as a contaminant on plasticware. We optimize our protein production processes to ensure the lowest possible levels of endotoxin contamination. Our endotoxin pass criteria are set at the industry leading <0.1 EU per ug protein and we aim for <0.01 EU per ug protein. Endotoxin levels in our proteins are determined by an external expert microbiological testing services provider. Example data from Qk017 batch #011
Recombinant GDF15 protein is very poorly soluble in physiological solutions. Please follow our handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing our recombinant human GDF15 protein at very low pH before dilution in cell culture media or your final working solution. Low pH will also assist in maintaining the correct disulphide structure of the protein by minimizing disulphide bond exchange reactions.
- Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilized cytokines in 10 mM HCl (1:1000 dilution of concentrated HCl) while keeping the protein concentration at 50 µg/ml or above, in order to avoid loss by adsorption to plasticware.
- To ensure you recover all of the protein, let the sample sit for a few minutes with the solubilization buffer at room temperature and pipette gently up and down (avoid foaming).
- Rinse the tube with some more 10 mM HCl and pool with the rest.
- The protein is tolerant of some freeze and thaw cycles, but as always with proteins, it is better to aliquot and stored frozen.
- Our protein are supplied carrier-protein free. If compatible with your work, add carrier protein of your choice, such as BSA, HSA or gelatin, to further minimize loss by adsorption.
- Store at -80°C for long-term storage or at -20°C for short-term storage.
Every effort is made to ensure samples are sterile; however, we recommend sterile filtering after dilution in media or the final working solution
We check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so you can rely on your calculated dilution.
Recovery: protein quantitation
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.
Absorbance at 280 nm: average 0.13
Recovered concentration: 0.13 cm-1 x 10 / 1.17 cm-1 mg ml-1 = 1.1 mg / ml
Recovery: 110% (>100% due to routine 10% over-fill of vials during aliquoting)
The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. This was diluted 1:10 and the UV spectrum 340-220 nm. Concentration was calculated using extinction coefficient at 280 nm.