Nonaffinity Purification of Recombinant gp120 for Use in AIDS Vaccine Development

Carl J. Scandella, Jaleh Kilpatrick, William Lidster, Charles Parker, John P. Moore, Gregory K. Moore, Kimberly A. Mann, Peter Brown, Stephen Coates, Barbara Chapman, Frank R. Masiarz, Kathelyn S. Steimer, Nancy L. Haigwood

    Research output: Contribution to journalArticle

    42 Scopus citations

    Abstract

    The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gelfiltration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd ˜2 x 10-9 M).

    Original languageEnglish (US)
    Pages (from-to)1233-1244
    Number of pages12
    JournalAIDS Research and Human Retroviruses
    Volume9
    Issue number12
    DOIs
    StatePublished - Dec 1993

    ASJC Scopus subject areas

    • Immunology
    • Virology
    • Infectious Diseases

    Fingerprint Dive into the research topics of 'Nonaffinity Purification of Recombinant gp120 for Use in AIDS Vaccine Development'. Together they form a unique fingerprint.

  • Cite this

    Scandella, C. J., Kilpatrick, J., Lidster, W., Parker, C., Moore, J. P., Moore, G. K., Mann, K. A., Brown, P., Coates, S., Chapman, B., Masiarz, F. R., Steimer, K. S., & Haigwood, N. L. (1993). Nonaffinity Purification of Recombinant gp120 for Use in AIDS Vaccine Development. AIDS Research and Human Retroviruses, 9(12), 1233-1244. https://doi.org/10.1089/aid.1993.9.1233