title: Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160.
abstract: The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.
authors.affiliation: Laboratory of Molecular Medicine, The Children's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02215, USA.
authors.forename: B
authors.initials: B
authors.lastname: Chen
authors.forename: G
authors.initials: G
authors.lastname: Zhou
authors.forename: M
authors.initials: M
authors.lastname: Kim
authors.forename: Y
authors.initials: Y
authors.lastname: Chishti
authors.forename: R E
authors.initials: RE
authors.lastname: Hussey
authors.forename: B
authors.initials: B
authors.lastname: Ely
authors.forename: J J
authors.initials: JJ
authors.lastname: Skehel
authors.forename: E L
authors.initials: EL
authors.lastname: Reinherz
authors.forename: S C
authors.initials: SC
authors.lastname: Harrison
authors.forename: D C
authors.initials: DC
authors.lastname: Wiley
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D015686:Gene Products, env / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D007313:Insecta; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014461:Ultracentrifugation
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D003432:Cross-Linking Reagents; D015686:Gene Products, env; C087575:SIV envelope glycoprotein gp160