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Recombinant human Tat-Hsp70-2: A tool for neuroprotection.
Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano.
Afiliación
  • Cappelletti P; Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy; The Protein Factory Research Center, Politecnico of Milano and University of Insubria, Via Mancinelli 7, 20131 Milano, Italy. Electronic address: pamela.cappelletti@uninsubria.it.
  • Binda E; Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy; The Protein Factory Research Center, Politecnico of Milano and University of Insubria, Via Mancinelli 7, 20131 Milano, Italy.
  • Tunesi M; Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico of Milano, p.zza Leonardo da Vinci 32, 20133 Milano, Italy; Unità di Ricerca Consorzio INSTM, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133 Milano, Italy.
  • Albani D; IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy.
  • Giordano C; The Protein Factory Research Center, Politecnico of Milano and University of Insubria, Via Mancinelli 7, 20131 Milano, Italy; Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico of Milano, p.zza Leonardo da Vinci 32, 20133 Milano, Italy; Unità di Ricerca Consorzio
  • Molla G; Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy; The Protein Factory Research Center, Politecnico of Milano and University of Insubria, Via Mancinelli 7, 20131 Milano, Italy.
  • Pollegioni L; Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy; The Protein Factory Research Center, Politecnico of Milano and University of Insubria, Via Mancinelli 7, 20131 Milano, Italy.
Protein Expr Purif ; 138: 18-24, 2017 Oct.
Article en En | MEDLINE | ID: mdl-27405095
Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.
Asunto(s)
Adenosina Trifosfatasas/biosíntesis; Proteínas HSP70 de Choque Térmico/biosíntesis; Fármacos Neuroprotectores/metabolismo; Proteínas Recombinantes de Fusión/biosíntesis; Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis; Adenosina Trifosfatasas/genética; Adenosina Trifosfatasas/aislamiento & purificación; Adenosina Trifosfatasas/farmacología; Línea Celular Tumoral; Supervivencia Celular/efectos de los fármacos; Clonación Molecular; Escherichia coli/genética; Escherichia coli/metabolismo; Expresión Génica; Proteínas HSP70 de Choque Térmico/genética; Proteínas HSP70 de Choque Térmico/aislamiento & purificación; Proteínas HSP70 de Choque Térmico/farmacología; Humanos; Peróxido de Hidrógeno/antagonistas & inhibidores; Peróxido de Hidrógeno/farmacología; Cuerpos de Inclusión/química; Neuronas/citología; Neuronas/efectos de los fármacos; Neuronas/metabolismo; Fármacos Neuroprotectores/aislamiento & purificación; Fármacos Neuroprotectores/farmacología; Estrés Oxidativo; Oxidopamina/antagonistas & inhibidores; Oxidopamina/farmacología; Pliegue de Proteína; Proteínas Recombinantes de Fusión/genética; Proteínas Recombinantes de Fusión/aislamiento & purificación; Proteínas Recombinantes de Fusión/farmacología; Solubilidad; Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética; Productos del Gen tat del Virus de la Inmunodeficiencia Humana/aislamiento & purificación; Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Adenosina Trifosfatasas / Fármacos Neuroprotectores / Proteínas HSP70 de Choque Térmico / Productos del Gen tat del Virus de la Inmunodeficiencia Humana Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2017 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Adenosina Trifosfatasas / Fármacos Neuroprotectores / Proteínas HSP70 de Choque Térmico / Productos del Gen tat del Virus de la Inmunodeficiencia Humana Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2017 Tipo del documento: Article Pais de publicación: Estados Unidos