Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide

• Published in: PloS one 2017-02, Vol.12 (2), p.e0172529
• Main Authors: Deb, Arpan, Johnson, William A, Kline, Alexander P, Scott, Boston J, Meador, Lydia R, Srinivas, Dustin, Martin-Garcia, Jose M, Dörner, Katerina, Borges, Chad R, Misra, Rajeev, Hogue, Brenda G, Fromme, Petra, Mor, Tsafrir S
• Format: Article
• Language: English
• Subjects: Amino acids; Aquaporins; Bacteria; Biology and life sciences; CD4 antigen; Cell membranes; Chromatography; Circular dichroism; Cloning, Molecular; Coding; Dichroism; Down-regulation; E coli; Erwinia carotovora; Escherichia coli; Gene Expression; Health aspects; HIV; Human immunodeficiency virus; Human Immunodeficiency Virus Proteins - genetics; Human Immunodeficiency Virus Proteins - metabolism; Humans; Immunotherapy; Inserts; Integral membrane proteins; Lentivirus; Life sciences; Light scattering; Membrane proteins; Membranes; Pathogenesis; Pectate lyase; Photon correlation spectroscopy; Physical Sciences; Progeny; Protein Folding; Protein Sorting Signals; Proteins; Receptor mechanisms; Receptors; Research and Analysis Methods; Retroviridae; Structural models; Structure-function relationships; Viral proteins; Viral Regulatory and Accessory Proteins - genetics; Viral Regulatory and Accessory Proteins - metabolism; Virology; Viruses; Vpu protein
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0172529

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.