title: Recombinant human Tat-Hsp70-2: A tool for neuroprotection.
abstract: 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.
authors.affiliation: 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.
authors.forename: Pamela
authors.initials: P
authors.lastname: Cappelletti
authors.affiliation: 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.
authors.forename: Elisa
authors.initials: E
authors.lastname: Binda
authors.affiliation: 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.
authors.forename: Marta
authors.initials: M
authors.lastname: Tunesi
authors.affiliation: IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: Albani
authors.affiliation: 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 INSTM, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133 Milano, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Giordano
authors.affiliation: 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.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.affiliation: 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.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002479:Inclusion Bodies / Q000737:chemistry; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D018384:Oxidative Stress; D016627:Oxidopamine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D018840:HSP70 Heat-Shock Proteins; C525621:HSPA1B protein, human; D018696:Neuroprotective Agents; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D016627:Oxidopamine; D006861:Hydrogen Peroxide; D000251:Adenosine Triphosphatases
keywords: Inclusion bodies; Neuroprotection; Protein production; Tat-proteins