Chick Endoglin: From cDNA to Interacting Partners

Endoglin, a transmembrane protein on endothelial cells, is required for angiogenesis. In humans and chick it is reported to be an auxiliary receptor in the TGFβ receptor complex containing receptor types I, II and auxiliary type III. Binding of TGFβ growth factors to their receptors regulates responses such as cell differentiation and migration. In this work we generated GST fusion proteins to confirm endoglin’s interacting partners in chick. To begin, overlapping cDNA clones were captured, assembled into full‐length cDNA and its consensus sequence determined (GenBank AY702002). The latter was used to design and generate four GST‐endoglin pGEX constructs each housing a portion of endoglin’s extracellular domain (ECD). Constructs were transformed into E. coli BL21 cells and induced to express fusion protein. Proteins of predicted sizes were detected by Western analysis. Expression of GST‐endoglin‐D encoding 80% of the ECD was optimized and recovered from bacterial sonicates. Fusion protein was initially purified on glutathione sepharose beads. Further purification by preparative PAGE recovered endoglin‐D protein in amounts sufficient for antisera production now in progress. GST‐endoglin‐D is also being used in a GST pulldown assay to recover its interacting partners from lysates of 3‐6 DIO chick embryos reported in the literature to contain the candidate proteins. Antibodies against TGFβ receptor types I, II and III and TFGβ −3 are being used to confirm these candidate partners. When available antibodies against chick endoglin will also be used. GST‐endoglin fusion proteins housing shorter regions of the endoglin ECD will be used to localize interacting regions of chick endoglin. FRCWA, SRA