Ligand-independent assembly of purified soluble magic roundabout (Robo4), a tumor-specific endothelial marker.
Magic roundabout (Robo4) is the fourth recently identified member of the roundabout receptor family. Robo4 is predominantly expressed in embryonic or tumor vascular endothelium and is considered important for vascular development and as a candidate tumor endothelial marker. Much remains unknown about the Robo4 molecule, however, such as its ligands, structure, and the details of its function. Thus, we aimed to establish an expression and purification method for obtaining soluble recombinant human Robo4 (shRobo4) and mouse Robo4 (smRobo4) for use in Robo4 characterization studies. In this work, we expressed the extracellular domain of hRobo4 and mRobo4 in mammalian 293F cells and purified them by two-step chromatography. Based on gel-filtration chromatography and Blue Native polyacrylamide gel electrophoresis, these purified proteins exist as multimers. The shRobo4 and smRobo4 we obtained will be useful in advanced studies to determine the importance of multimerization, identify the ligands, and elucidate the ligand-receptor interactions and Robo4-mediated signaling. The results of these studies will help to elucidate the role of Robo4 in angiogenesis and perhaps eventually contribute to the development of novel vessel-targeting therapies.
Yoshikawa
Mai
M
Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Mukai
Yohei
Y
Okada
Yoshiaki
Y
Yoshioka
Yasuo
Y
Tsunoda
Shin-Ichi
S
Tsutsumi
Yasuo
Y
Okada
Naoki
N
Aird
William C
WC
Doi
Takefumi
T
Nakagawa
Shinsaku
S
D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D009389:Neovascularization, Pathologic; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D000097905:Roundabout Proteins
D014408:Biomarkers, Tumor; D017931:DNA Primers; D008024:Ligands; D011956:Receptors, Cell Surface; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins