title: Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.
abstract: Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Arpan
authors.initials: A
authors.lastname: Deb
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: William A
authors.initials: WA
authors.lastname: Johnson
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Kline
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Boston J
authors.initials: BJ
authors.lastname: Scott
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Lydia R
authors.initials: LR
authors.lastname: Meador
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Dustin
authors.initials: D
authors.lastname: Srinivas
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Martin-Garcia
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Personal Diagnostics, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Chad R
authors.initials: CR
authors.lastname: Borges
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Misra
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Brenda G
authors.initials: BG
authors.lastname: Hogue
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.|The Biodesign Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Tsafrir S
authors.identifier: http://orcid.org/0000-0002-6123-6194
authors.initials: TS
authors.lastname: Mor
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017510:Protein Folding; D021382:Protein Sorting Signals; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D054298:Human Immunodeficiency Virus Proteins; D021382:Protein Sorting Signals; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins