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TGF-? contamination of purified recombinant GDF15.

ABSTRACT: Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-? superfamily. TGF-? itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-? in purified protein batches may influence the experimental results given that receptors for TGF-? are present. When we attempted to characterize possible receptors for the TGF-? superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-? activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-? and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results.

SUBMITTER: Olsen OE

PROVIDER: S-EPMC5697882 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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TGF-β contamination of purified recombinant GDF15.

Olsen Oddrun Elise OE Skjærvik Anette A Størdal Berit Fladvad BF Sundan Anders A Holien Toril T

PloS one 20171121 11

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in ...[more]

PMID: 29161287

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Project description:Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

Project description:BackgroundCardiac fibrosis refers to the abnormal accumulation of extracellular matrix in the heart, which leads to the formation of cardiac scars. It causes systolic and diastolic dysfunction, and ultimately leads to cardiac dysfunction and arrhythmia. TGF-β1 is an important regulatory factor involved in cardiac fibrosis. Studies have shown that the N-terminal latency associated peptide (LAP) must be removed before TGF-β1 is activated. We hypothesize that recombinant LAP may inhibit cardiac fibrosis induced by TGF-β1. To evaluate anti-cardiac fibrosis activity of recombinant LAP, an experimental study was carried out and is reported here.MethodsThe pET28a-LAP plasmid was constructed and transformed into E. coli C43 (DE3) competent cells. The recombinant LAP protein was purified by Ni affinity chromatography. The cells were treated with TGF-β1 at different concentrations for 24 h. The expression of α-SMA was detected by Western blot. RTCA was used to detect the effect of recombinant LAP on the proliferation of H9C2 cells induced by 10 ng/mL TGF-β1. To detect the effect of LAP on the expression of fibrosis-related proteins, H9C2 cells were treated with 10 ng/mL TGF-β1 for 24 h, then added 60 μg/mL recombinant LAP for 48 h. The LAP group was treated with 60 μg/mL recombinant LAP alone. The LAP pre-protection group was treated with 10 ng/mL TGF-β1 and 60 μg/mL recombinant LAP at the same time. Western blot and immunofluorescence were used to detect the expression of α-SMA, collagen I and fibronectin and p-Smad2.ResultsThe recombinant LAP was prokaryotic expressed and purified. 10 ng/mL was determined as the optimal working concentration of TGF-β1 to induce H9C2 cells fibrosis. RTCA results showed that 60 μg/mL LAP could effectively inhibit the proliferation of H9C2 cells induced by TGF-β1. Immunofluorescence results showed that compared with the control group, the fluorescence intensities of α-SMA, collagen I and FN increased significantly after TGF-β1 treatment. The fluorescence intensities in the TGF-β1+LAP group decreased significantly. Western blot results showed that 60 μg/mL LAP could inhibit the increase of α-SMA, collagen I and FN expression in H9C2 cells induced by TGF-β1. Compared with the control, the LAP alone group has no significant difference in α-SMA and p-Smad2 expression level. The expression of α-SMA and p-Smad2 in the TGF-β1 model group was significantly increased compared with the control group. Compared with the TGF-β1 group, both TGF-β1+LAP group and LAP pre-protection group significantly reduced the increase in α-SMA and p-Smad2 levels.ConclusionsRecombinant LAP was prokaryotic expressed and purified. The results showed that recombinant LAP can inhibit the cell proliferation and expression increase of α-SMA, collagen I, fibronectin and p-Smad2 in H9C2 cells induced by TGF-β1. These results suggested that recombinant LAP might inhibit TGF-β1-induced fibrosis of H9C2 cells through the TGF-β/Smad pathway.

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TGF-? contamination of purified recombinant GDF15.

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TGF-β contamination of purified recombinant GDF15.

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