title: Expression and characterization of recombinant canine IL-13 receptor alpha2 protein and its biological activity in vitro.
abstract: Our previous study showed that recombinant canine IL-13 (rcaIL-13) stimulated production of allergen-specific IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs. This has also been demonstrated using human IL-13 (huIL-13) and PBMC isolated from human allergy patients. The stimulatory activity of rcaIL-13 was specifically inhibited by a fusion protein of the extracellular domain of canine IL-13Ralpha2 and the Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). In this communication, we report the construction and expression of a non-fused recombinant extracellular domain of canine IL-13Ralpha2 (rcaIL-13Ralpha2) in an E. coli expression system. The E. coli expressed rcaIL-13Ralpha2 was isolated in inclusion bodies, then solubilized in buffer containing denaturants and reducing agents. After refolding and purification, the biological activity of rcaIL-13Ralpha2 was found in the monomer fraction resulting from gel filtration and ion exchange chromatographies. Biological activity of purified rcaIL-13Ralpha2 was demonstrated by the specific inhibition of rcaIL-13 activity in a TF-1 cell proliferation assay. Additionally, rcaIL-13Ralpha2 was found to be active in neutralizing rcaIL-13 induced upregulation of IgE mRNA levels in PBMCs of "high IgE" dogs, which have been bred to exhibit a predisposition for high IgE production and are used as a model for allergic asthma. The data confirm our previous report that the regulatory effects of IL-13 on IgE production in canine PBMCs are similar to those reported in humans. Thus, allergic dogs, such as the "high IgE" producing dogs, may be excellent models for research on IgE-mediated diseases in humans.
authors.affiliation: Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Tang
authors.forename: Tony
authors.initials: T
authors.lastname: Morales
authors.forename: Karen L
authors.initials: KL
authors.lastname: Boroughs
authors.forename: Kathy
authors.initials: K
authors.lastname: Cailles Lo-Keiser
authors.forename: Karen
authors.initials: K
authors.lastname: Sellins
authors.forename: Kim
authors.initials: K
authors.lastname: Stedman
authors.forename: Catherine
authors.initials: C
authors.lastname: McCall
authors.forename: Martin J
authors.initials: MJ
authors.lastname: McDermott
mesh_terms: D000485:Allergens; D000818:Animals; D001249:Asthma / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000096:biosynthesis; D066298:In Vitro Techniques; D018793:Interleukin-13 / Q000494:pharmacology; D053661:Interleukin-13 Receptor alpha1 Subunit; D007963:Leukocytes, Mononuclear / Q000276:immunology; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D053660:Receptors, Interleukin-13; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D000485:Allergens; D018076:DNA, Complementary; C508181:IL13RA1 protein, human; D018793:Interleukin-13; D053661:Interleukin-13 Receptor alpha1 Subunit; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053660:Receptors, Interleukin-13; D011994:Recombinant Proteins; D007073:Immunoglobulin E