Twisted gastrulation polypeptides and uses thereof
a gastrulation polypeptide and polypeptide technology, applied in the direction of peptide/protein ingredients, peptide sources, extracellular fluid disorders, etc., can solve the problems of poor therapeutic response, many individuals are refractory to even high doses, and the effect of epo is not uniformly effectiv
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n of Human TWSG-Fc Fusion Protein
[0198]Applicants constructed a soluble TWSG fusion protein (TWSG-Fc) in which full-length human TWSG (FIG. 1, SEQ ID NO: 8) was attached at its C-terminus to a human IgG1 Fc domain (SEQ ID NO: 3) with a minimal linker. TWSG-Fc (FIG. 2, SEQ ID NO: 9) was initially expressed by transient transfection in COS cells, as were N-glycosylation variants of TWSG-Fc (see below). In brief, COS cells (ATCC®) were transfected overnight with plasmid encoding TWSG-Fc using FuGENE® 6 transfection reagent (Promega). The next day, cells were washed with phosphate-buffered saline, and serum-free medium was added. After incubation for 72 h, the COS-conditioned medium was harvested, filtered, and loaded on a MabSelect SuRe column (GE Healthcare, UK). Fusion protein was eluted with 0.1 M glycine (pH 3.0), and the eluted fractions were immediately neutralized by addition 1 M Tris (pH 8.0) in a 1:10 ratio. Protein was quantitated using a NanoDrop™ spectrophotometer (Thermo F...
example 2
nding to Murine TWSG and Human TWSG-Fc
[0203]Previous studies have determined that TWSG, or its nonmammalian homolog Tsg, binds with high affinity to BMP2, BMP4, and BMP7 (Oelgeschläger et al., 2000, Nature 405:757-763; Scott et al., 2001, Nature 410:475-478; Chang et al., 2001, Nature 410:483-487). Since these studies have not systematically evaluated TWSG (or Tsg) binding to other TGFβ superfamily ligands, Applicants used surface plasmon resonance to investigate and characterize such binding. In an initial qualitative screen, recombinant murine TWSG (mTWSG; R&D Systems, Minneapolis, Minn.) was covalently immobilized on a BIACORE™ CMS chip, and more than 30 ligands generated in-house or obtained from R&D Systems were injected individually over the captured mTWSG to characterize their degree of binding at room temperature. Based on the results of this screen, Applicants subjected selected ligands to quantitative characterization of binding to human TWSG fusion protein at physiologic ...
example 3
n of Ligand Signaling by TWSG-Fc in Cell-Based Assays
[0206]Reporter gene assays were used to determine the ability of human TWSG-Fc to inhibit signaling by BMP2, BMP4, BMP6, BMP7, BMP9, and BMP10. These assays are based on human glioblastoma (T98G) or hepatocellular carcinoma (HepG2) cell lines transfected with a pGL3 BRE (BMP response element) reporter plasmid (Korchynskyi et al., 2002, J Biol Chem 277: 4883-4891) as well as a Renilla reporter plasmid (pRLCMV) to control for transfection efficiency. BMP response elements from the Id1 promoter are present in the promoter of the pGL3 BRE reporter plasmid, so this vector is of general use for factors signaling through Smad1 and Smad5.
[0207]On the first day of the assay, T98G cells (ATCC®: CRL_1690™) or HepG2 cells (ATCC®: HB-8065™) were distributed in 48-well plates at 8.5×104 cells per well or 12.5×104 cells per well, respectively. On the second day, a solution containing 10 μg pGL3 BRE, 100 ng pRLCMV, 30 μl Fugene HD (Roche Applied ...
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