Agents for reducing the activity of gdf15
A technology of X1X2X3X4X5X6X7X8X9, binding agent, applied in the direction of anti-animal/human immunoglobulin, animal/human protein, antibody mimic/scaffold, etc., can solve the problem of non-existence and difficult to reverse cachexia
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Embodiment 1-
[0301] Example 1 - Tandem Affinity Purification - Mass Spectrometry
[0302] We designed a protein-protein interaction capture assay using a panel of different proteins with the same affinity tag and included GDF15 to screen for interacting proteins. GDF15 was chosen for inclusion in the panel due to its important role in human pathology, including various cancers, and the lack of scientific literature on interacting proteins and receptors.
[0303] Design and construction of multiple bait proteins
[0304] Bioinformatics (SSPRO, RASMOL) analyzes were performed to rule out the presence of hydrophobic amino acids in the baits and to avoid disruption of known secondary structure elements. The bait insert was PCR amplified from plasmid DNA and inserted into a eukaryotic expression vector allowing gentamicin selection.
[0305] Cells were transfected with expression vectors, and overexpressed stable cell lines were generated in parallel against a panel of bait proteins, includ...
Embodiment 2
[0317] Example 2 - Purification of QRFPR-containing exosomes from conditioned media.
[0318] Exosome Isolation
[0319] Conditioned cell cultures were sequentially centrifuged at 3,000 x g for 5 min to pellet the cells and then at 10,000 x g for 10 min to further remove cells and cell debris. The supernatant was then filtered through a 0.45 μm filter and finally pelleted at 100,000 x g for 2 h at 4°C. The pelleted exosomes were resuspended in PBS and protease inhibitors (completemini, Roche) added. Samples were then prepared for further analysis as described elsewhere.
[0320] Exosome Identification by Electron Microscopy
[0321] Clumped exosomes were precipitated on 20-mesh polymethylvinyl acetate / carbon-coated copper grids (polysciences, Inc.), and then detected by transmission electron microscopy at 80 kV in a Technai G2 (FEI). (TEM) for analysis ( figure 1 A).
[0322] Western blot identification of QRFPR-positive exosomes
[0323] The material from the ultrac...
Embodiment 3
[0324] Example 3 - Detection of GDF15-QRFPR on the cell surface
[0325] To investigate the presence of the complex between GDF15 and QRFPR on the cell surface, we analyzed MCF-7 treated with sulindac sulfide (an NSAID to increase the amount of low endogenous GDF15) using in situ PLA cells to examine the interaction between GDF15 and QRFPR. We compared siRNA against QRFPR ( figure 2 B) or control siRNA wrapped with sequence ( figure 2 A) Levels of proximity events between GDF15 and QRFPR in treated MCF-7 cells. We detected reduced GDF15-QRFPR levels in QRFPR siRNA-treated cells, confirming the specificity of the antibody and the presence of the receptor in this cell line. Cells were serum starved prior to GDF15 treatment.
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