title: Recombinant expression, purification and dimerization of the neurotrophic growth factor Artemin for in vitro and in vivo use.
abstract: Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain paradigms. In this paper, we describe the development of an efficient method for the recombinant bacterial production of large quantities of highly pure, biologically active ARTN for in vitro and in vivo studies. Using Escherichia coli expression of an NH(2)-terminal SUMO-Artemin fusion protein and subsequent refolding from inclusion bodies followed by cleavage of the SUMO fusion part, mature Artemin with a native NH(2)-terminal amino acid sequence was obtained at high purity (>99%). Experiments using the reducing agent dithiothreitol (DTT) demonstrated that the intermolecular disulphide bridge in the cysteine knot is dispensable for dimerization of stable ARTN monomers. Our production method could facilitate in vitro and in vivo experimentation for the possible development of Artemin as a therapeutic agent for neuropathic pain.
authors: Department of Assay Development & Target Validation, C.R.E.A. Te, Janssen Research & Development, B-2340 Beerse, Belgium.
authors: Wouter
authors: W
authors: Bruinzeel
authors: Department of Assay Development & Target Validation, C.R.E.A. Te, Janssen Research & Development, B-2340 Beerse, Belgium. Electronic address: smasure@its.jnj.com.
authors: Stefan
authors: S
authors: Masure
mesh_terms: D003001:Cloning, Molecular; D003956:Dialysis; D004229:Dithiothreitol; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D025842:SUMO-1 Protein
chemical_list: C490886:ARTN protein, human; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D004229:Dithiothreitol