title: Exceptional stability of artemin neurotrophic factor dimers: effects of temperature, pH, buffer and storage conditions on protein integrity and activity.
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. We have developed a robust and sensitive cellular assay to measure ARTN biological activity. Using recombinant Artemin produced in Escherichia coli bacteria together with this specific assay, we demonstrate that ARTN is an exceptionally stable polypeptide. Multiple freeze-thaw cycles, incubation at elevated temperatures (up to 90 °C) for 0.5 h, prolonged storage at 4 °C, and exposure to conditions of different pH, salt concentration, and additives had no measurable effect on the biological activity of ARTN. In some of the tested conditions, partial removal of nine NH(2)-terminal amino acids of the ARTN protein occurred, but this truncation had no important effect on the ARTN signaling response. Consequently, we postulate that formulation and storage for in vivo testing of ARTN in neuropathic pain paradigms in animals and humans should be straightforward.
authors: Department of Assay Development & Target Validation, C.R.E.A.Te, Janssen Research & Development, Beerse, Belgium.
authors: Wouter
authors: W
authors: Bruinzeel
authors: Stefan
authors: S
authors: Masure
mesh_terms: D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D019281:Dimerization; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D055550:Protein Stability; D013696:Temperature
chemical_list: C490886:ARTN protein, human; D009419:Nerve Tissue Proteins