title: Expression and purification of soluble HIV-2 viral protein R (Vpr) using a sandwich-fusion protein strategy.
abstract: Viral accessory proteins of the human immunodeficiency virus (HIV), including virus protein R (Vpr), are crucial for the efficient replication of the virus in the host organism. While functional data are available for HIV-1 Vpr, there is a paucity of data describing the function and structure of HIV-2 Vpr. In this report, the construction of a His6-MBP-intein1-Vpr-intein2-Cyt b5-His6 fusion protein is presented. Unlike previous research efforts where only microgram quantities of HIV-1 Vpr could be produced, this construct enabled soluble milligram yields via an Escherichia coli over-expression system. Straightforward protein purification of HIV-2 Vpr was achieved by standard chromatography routines and autocatalytic intein cleavage. Preliminary structural studies by circular dichroism (CD) and NMR spectroscopy revealed that the protein is stable in the presence of micellar concentrations of the detergent DPC and adopts an α-helix secondary structure.
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems (ICS-6), Wilhelm-Johnen-Straße, 52425 Jülich, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Hänel
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems (ICS-6), Wilhelm-Johnen-Straße, 52425 Jülich, Germany.
authors.forename: Luis
authors.initials: L
authors.lastname: Möckel
authors.affiliation: Westfälische Hochschule, Molekulare Biologie, August-Schmidt-Ring 10, 45665 Recklinghausen, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Brummel
authors.affiliation: Westfälische Hochschule, Molekulare Biologie, August-Schmidt-Ring 10, 45665 Recklinghausen, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Peiris
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems (ICS-6), Wilhelm-Johnen-Straße, 52425 Jülich, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Hartmann
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems (ICS-6), Wilhelm-Johnen-Straße, 52425 Jülich, Germany.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Dingley
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems (ICS-6), Wilhelm-Johnen-Straße, 52425 Jülich, Germany; Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Universitätsstraße 1, 40225 Düsseldorf, Germany. Electronic address: dieter.willbold@hhu.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.affiliation: Westfälische Hochschule, Molekulare Biologie, August-Schmidt-Ring 10, 45665 Recklinghausen, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Loidl-Stahlhofen
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514913:vpr protein, Human immunodeficiency virus 2; D010767:Phosphorylcholine; C028810:dodecylphosphocholine
keywords: DPC; HIV accessory proteins; HIV-2 Vpr; Intein; NMR; Sandwich fusion protein