Recombinant human Tat-Hsp70-2: A tool for neuroprotection

Pamela Cappelletti; Elisa Binda; Marta Tunesi; Diego Albani; Carmen Giordano; Gianluca Molla; Loredano Pollegioni

doi:10.1016/j.pep.2016.07.005

Recombinant human Tat-Hsp70-2: A tool for neuroprotection

Protein Expr Purif. 2017 Oct:138:18-24. doi: 10.1016/j.pep.2016.07.005. Epub 2016 Jul 9.

Authors

Pamela Cappelletti¹, Elisa Binda², Marta Tunesi³, Diego Albani⁴, Carmen Giordano⁵, Gianluca Molla², Loredano Pollegioni²

Affiliations

¹ 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.
² 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.
³ 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.
⁴ IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy.
⁵ 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.

PMID: 27405095
DOI: 10.1016/j.pep.2016.07.005

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.

Keywords: Inclusion bodies; Neuroprotection; Protein production; Tat-proteins.

MeSH terms

Adenosine Triphosphatases / biosynthesis*
Adenosine Triphosphatases / genetics
Adenosine Triphosphatases / isolation & purification
Adenosine Triphosphatases / pharmacology
Cell Line, Tumor
Cell Survival / drug effects
Cloning, Molecular
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
HSP70 Heat-Shock Proteins / biosynthesis*
HSP70 Heat-Shock Proteins / genetics
HSP70 Heat-Shock Proteins / isolation & purification
HSP70 Heat-Shock Proteins / pharmacology
Humans
Hydrogen Peroxide / antagonists & inhibitors
Hydrogen Peroxide / pharmacology
Inclusion Bodies / chemistry
Neurons / cytology
Neurons / drug effects
Neurons / metabolism
Neuroprotective Agents / isolation & purification
Neuroprotective Agents / metabolism*
Neuroprotective Agents / pharmacology
Oxidative Stress
Oxidopamine / antagonists & inhibitors
Oxidopamine / pharmacology
Protein Folding
Recombinant Fusion Proteins / biosynthesis*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / isolation & purification
Recombinant Fusion Proteins / pharmacology
Solubility
tat Gene Products, Human Immunodeficiency Virus / biosynthesis*
tat Gene Products, Human Immunodeficiency Virus / genetics
tat Gene Products, Human Immunodeficiency Virus / isolation & purification
tat Gene Products, Human Immunodeficiency Virus / pharmacology

Substances

HSP70 Heat-Shock Proteins
HSPA1B protein, human
Neuroprotective Agents
Recombinant Fusion Proteins
tat Gene Products, Human Immunodeficiency Virus
Oxidopamine
Hydrogen Peroxide
Adenosine Triphosphatases