High level production and one-step purification of biologically active ectodysplasin A1 and A2 immunoadhesins using the baculovirus/insect cell expression system.
Ectodysplasin A (EDA) is a ligand of the tumor necrosis factor (TNF) family that has been shown to play a crucial role in ectodermal differentiation. Mutations of the syntenic ectodysplasin A gene (Eda) are responsible for Tabby (Ta) phenotype in mice and human X-linked hypohidrotic ectodermal dysplasia (XLHED). EDA-A1 and EDA-A2 are the two main splice variants of Eda, which differ from each other in only two amino acid residues and engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. We have used the baculovirus/insect cell system to express the recombinant EDA proteins fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Immunoadhesins (4.5-4.7 mg/L) from crude supernatant could be purified to near homogeneity by using rProtein A affinity chromatography. The purified EDA immunoadhesins were endowed with ligand-binding activity as they could bind EDAR or XEDAR on the surface of 293T cells that had been transiently transfected with the corresponding plasmids. Functional activities of EDA immunoadhesins were demonstrated by their ability to activate the NF-kappaB pathway in cells expressing their cognate receptors. These results open up the possibility of obtaining large amounts of purified EDA proteins to investigate EDAR/XEDAR related signaling pathways and for the treatment of patients with X-linked hypohidrotic ectodermal dysplasia.
Chang
Bingsheng
B
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA.
Chaudhary
Preet M
PM
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D053331:Ectodysplasins; D017930:Genes, Reporter; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007313:Insecta; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C506018:EDA protein, human; D053331:Ectodysplasins; C506019:Eda protein, mouse; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins