title: Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity.
abstract: Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, WA 98195, USA. wguo@wanprc.org
authors.forename: Wenjin
authors.initials: W
authors.lastname: Guo
authors.forename: Brad
authors.initials: B
authors.lastname: Cleveland
authors.forename: Thaddeus M
authors.initials: TM
authors.lastname: Davenport
authors.forename: Kelly K
authors.initials: KK
authors.lastname: Lee
authors.forename: Shiu-Lok
authors.initials: SL
authors.lastname: Hu
mesh_terms: D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019302:HIV Envelope Protein gp160 / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000378:metabolism*
chemical_list: D003902:Detergents; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D009930:Organic Chemicals; D011498:Protein Precursors; D011994:Recombinant Proteins; C041772:empigen BB