Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule

D. E. McCallus, K. E. Ugen, A. I. Sato, W. V. Williams, D. B. Weiner

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The CD4 protein expressed on helper T lymphocytes is a restriction element for major histocompatibility class II immune responses. This molecule is also used by the human immunodeficiency virus as its specific cellular receptor facilitating binding of virus to cells. As soluble forms of CD4 inhibit HIV infection in tissue culture, attention has focused on this molecule. Bacterially produced CD4 would facilitate studies of the biology of the CD4 molecule. However, bacterially expressed CD4 must be refolded for assumption of its interaction with conformationally dependant anti-CD4 monoclonal antibodies as well as the HIV-1 envelope protein gp120. We report here the engineering of an external domain construct of the CD4 gene into a novel expression vector containing the nucleotide sequence encoding the pelB leader peptide of Erwinia carotovara (pDAB(L)), to facilitate correct folding of CD4 in bacteria. Monoclonal antibodies specific for important conformational epitopes of the CD4 molecule were able to bind bacterial colonies containing the pDAB(L)/CD4 vector but not colonies with vector alone. Importantly, recombinant gp120 produced in baculovirus bound specifically to bacterial colonies expressing the CD4 recombinant molecule. This system presents a simple screening mechanism for molecules that bind to the external domain of the CD4 glycoprotein. Vectors such as pDAB(L) will also facilitate the production of large amounts of biologically active proteins in bacteria.

Original languageEnglish (US)
Pages (from-to)163-172
Number of pages10
JournalViral Immunology
Volume5
Issue number2
DOIs
StatePublished - 1992
Externally publishedYes

ASJC Scopus subject areas

  • Immunology
  • Molecular Medicine
  • Virology

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