Cloning and expression of an HIV-1 specific single-chain Fv region fused to Escherichia coli alkaline phosphatase.
We have constructed a single-chain Fv fragment representing the variable domain of the human monoclonal antibody 3D6, binding specifically to HIV-1 gp41. This gene was fused to the coding region of E. coli alkaline phosphatase (EcPhoA) and expressed in E. coli. The EcPhoA signal peptide was used to direct the recombinant fusion protein to the periplasmic space of the bacteria, from where it was purified by hydrophobic interaction chromatography and gel filtration followed by antigen-affinity chromatography using a synthetic HIV-1 peptide as ligand. The purified fusion protein was bifunctional, showing both phosphatase activity as well as antigen-binding specificity identical to that of the original antibody.
Kohl
J
J
Institut für Angewandte Mikrobiologie, Universität für Bodenkultur, Nussdorfer Lände 11, Vienna, Austria.
Rüker
F
F
Himmler
G
G
Razazzi
E
E
Katinger
H
H
D000469:Alkaline Phosphatase / Q000235:genetics*; D000914:Antibodies, Viral / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D004247:DNA; D000469:Alkaline Phosphatase