title: Isolation and characterization of recombinant murine Wnt3a.
abstract: 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.
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Witkowski
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Aparna
authors.initials: A
authors.lastname: Krishnamoorthy
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Betty
authors.initials: B
authors.lastname: Su
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Beckstead
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States. Electronic address: rryan@chori.org.
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002793:Cholic Acids / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004396:Coloring Agents / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004331:Drosophila melanogaster / Q000166:cytology; D008024:Ligands; D051379:Mice; D059748:Proteolysis / Q000187:drug effects; D012995:Solubility; D013917:Thrombin / Q000494:pharmacology; D060509:Wnt3A Protein / Q000302:isolation & purification* / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
chemical_list: D002793:Cholic Acids; D004396:Coloring Agents; D017077:Culture Media, Conditioned; D008024:Ligands; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D013917:Thrombin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate
keywords: Chromatography; Drosophila S2 cells; L cells; Methyl-ß-cyclodextrin; Murine Wnt3a; Thrombin; ß-Catenin