Purification and Wnt-inhibitory activities of secreted frizzled-related proteins.
Recombinant expression of secreted Frizzled-related proteins (sFRPs) in mammalian expression systems is a convenient source of these proteins for biological studies. Yields of protein vary; screening of clonal lines for high expression is usually worthwhile. Heparin affinity chromatography is an easy step that provides a major enrichment, particularly for sFRP-1 and sFRP-2. Alternatively, sFRP derivatives tagged with poly-histidine at their carboxyl termini are functional and can be readily isolated by chelating chromatography. Once purified, the proteins are stable indefinitely if stored frozen and they tolerate multiple rounds of freeze-thawing. Pre-incubation of Wnt samples with sFRP protein for 30 min at 37 degrees C is sufficient to inhibit Wnt activity in various assays. The concentration of sFRP required to block Wnt signaling should be determined empirically, as it will vary with the Wnt preparation and cellular context.
Wolf
Vladimir
V
National Cancer Institute, Bethesda, MD, USA.
Endo
Yoshimi
Y
Rubin
Jeffrey S
JS
D000818:Animals; D002460:Cell Line; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D000099274:Frizzled-Related Proteins; D000094742:Secreted Frizzled-Related Proteins
D017077:Culture Media, Conditioned; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000099274:Frizzled-Related Proteins; C109618:SFRP2 protein, human; D000094742:Secreted Frizzled-Related Proteins