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 NH2-terminal SUMO-Artemin fusion protein and subsequent refolding from inclusion bodies followed by cleavage of the SUMO fusion part, mature Artemin with a native NH2-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.