Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule.
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 dependent 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 (pDABL), 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 pDABL/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 pDABL will also facilitate the production of large amounts of biologically active proteins in bacteria.
McCallus
D E
DE
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania.
Ugen
K E
KE
Sato
A I
AI
Williams
W V
WV
Weiner
D B
DB
D001483:Base Sequence; D015139:Blotting, Southern; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016973:Pectobacterium carotovorum / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes