title: Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41.
abstract: The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼ 1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, 85287-1604; The Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona, 85287.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Gong
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kessans
authors.forename: Lusheng
authors.initials: L
authors.lastname: Song
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.forename: Ho-Hsien
authors.initials: HH
authors.lastname: Lee
authors.forename: Lydia R
authors.initials: LR
authors.lastname: Meador
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
authors.forename: Brenda G
authors.initials: BG
authors.lastname: Hogue
authors.forename: Tsafrir S
authors.initials: TS
authors.lastname: Mor
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012542:Scattering, Radiation; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015700:HIV Envelope Protein gp41; D011994:Recombinant Proteins
keywords: HIV-1; dynamic light scattering; gp41; membrane proximal region; surface plasmon resonance; transmembrane domain