Isolation and characterization of recombinant murine Wnt3a.
Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects.
['Witkowski|Andrzej|A|', 'Krishnamoorthy|Aparna|A|', 'Su|Betty|B|', 'Beckstead|Jennifer A|JA|', 'Ryan|Robert O|RO|']
D000818:Animals D001671:Biochemistry D002793:Cholic Acids D002846:Chromatography, Affinity D002850:Chromatography, Gel D004396:Coloring Agents D017077:Culture Media, Conditioned D004331:Drosophila melanogaster D008024:Ligands D051379:Mice D059748:Proteolysis D012995:Solubility D013917:Thrombin D060509:Wnt3A Protein D051176:beta Catenin
Murine Wnt3a Drosophila S2 cells Chromatography Thrombin Methyl-ß-cyclodextrin L cells ß-Catenin