Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development

C. J. Scandella, J. Kilpatrick, W. Lidster, C. Parker, J. P. Moore, G. K. Moore, K. A. Mann, P. Brown, S. Coates, B. Chapman, F. R. Masiarz, K. S. Steimer, Nancy Haigwood

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120(SF2)) 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. rgp120(SF2) exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120(SF2) 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. rgp120(SF2) 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 rgp120(SF2) forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (K(d) -2 x 10-9 M).

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

Fingerprint

AIDS Vaccines
HIV
Molecular Weight
Glycoproteins
High Pressure Liquid Chromatography
Monoclonal Antibodies
CD4 Antigens
Peptides
Cricetulus
Neutralizing Antibodies
Gel Chromatography
HIV-1
Epitopes
Ovary
Buffers
Gels
Viruses
Cell Line
Serum
Genes

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Scandella, C. J., Kilpatrick, J., Lidster, W., Parker, C., Moore, J. P., Moore, G. K., ... Haigwood, N. (1993). Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. AIDS Research and Human Retroviruses, 9(12), 1233-1244.

Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. / 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, Nancy.

In: AIDS Research and Human Retroviruses, Vol. 9, No. 12, 1993, p. 1233-1244.

Research output: Contribution to journalArticle

Scandella, CJ, Kilpatrick, J, Lidster, W, Parker, C, Moore, JP, Moore, GK, Mann, KA, Brown, P, Coates, S, Chapman, B, Masiarz, FR, Steimer, KS & Haigwood, N 1993, 'Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development', AIDS Research and Human Retroviruses, vol. 9, no. 12, pp. 1233-1244.
Scandella CJ, Kilpatrick J, Lidster W, Parker C, Moore JP, Moore GK et al. Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. AIDS Research and Human Retroviruses. 1993;9(12):1233-1244.
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, Nancy. / Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. In: AIDS Research and Human Retroviruses. 1993 ; Vol. 9, No. 12. pp. 1233-1244.
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