title: Expression, purification, and characterization of recombinant HIV gp140. The gp41 ectodomain of HIV or simian immunodeficiency virus is sufficient to maintain the retroviral envelope glycoprotein as a trimer.
abstract: Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.
authors.affiliation: Laboratory of Immunobiology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
authors.forename: C W
authors.initials: CW
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Chishti
authors.forename: R E
authors.initials: RE
authors.lastname: Hussey
authors.forename: E L
authors.initials: EL
authors.lastname: Reinherz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D015686:Gene Products, env / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006031:Glycosylation; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015497:HIV-1 / Q000737:chemistry / Q000472:pathogenicity; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*; D012191:Retroviridae Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015302:Simian Immunodeficiency Virus / Q000737:chemistry / Q000472:pathogenicity; D054299:env Gene Products, Human Immunodeficiency Virus
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015686:Gene Products, env; D015700:HIV Envelope Protein gp41; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; C082614:SIV envelope protein gp41; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1