title: In vitro production of an active neurotrophic factor, neuregulin-1: qualitative comparison of different cell-free translation systems.
abstract: The individual biological activities of many neurotrophic factors and their variants, which are produced by alternative splicing and proteolytic processing, often remain to be characterized. Bacterial protein production combined with protein refolding and purification is a conventional procedure to obtain active neurotrophic factors; however, the procedure is time consuming and appropriate protein refolding in vitro is sometimes unpredictable. Here we examined three distinct cell-free translation systems: reticulocyte lysate, Hela cell lysate and wheat germ extract, which may allow us to produce biologically active factors in a single tube. Taking type-I neuregulin-1 beta3 as an example, we produced neuregulin-1 protein from its mRNAs flanked by Cap nucleotide and/or internal ribosome entry site (IRES) and compared the yields and biological activity of translation products from these systems. The protein yield from IRES+ mRNA was highest in the Hela cell-free system, while background translation was lowest in the wheat germ system. The biological activity of both translation products was modest or negligible, however. Neuregulin-1 protein was produced in reticulocyte lysate at yields of 19 pmol/mL (~500 ng/mL); furthermore, it was potent at phosphorylating ErbB4 receptor and able to bind to heparin sulfate. These results demonstrate that the reticulocyte lysate translation system produces active neurotrophic factors in vitro and is useful for radiolabeling or preliminary assessment of novel neurotrophic factors and their variants.
authors.affiliation: Department of Molecular Neurobiology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Niigata 951-8585, Japan.
authors.forename: Ran
authors.initials: R
authors.lastname: Wang
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Iwakura
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Araki
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Sotoyama
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takei
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nawa
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics*; D020890:Neuregulin-1 / Q000096:biosynthesis* / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology*; D058849:Protein Refolding; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D014909:Wheat Germ Agglutinins / Q000737:chemistry
chemical_list: D009414:Nerve Growth Factors; D020890:Neuregulin-1; D014909:Wheat Germ Agglutinins