Production of an immunoenzymatic tracer combining a scFv and the acetylcholinesterase of Bungarus fasciatus by genetic recombination.
We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.
Choumet
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V
Unité des Venins, Institut Pasteur, Paris, France.
Cousin
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X
Bon
C
C
D000110:Acetylcholinesterase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017838:Bungarus; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004546:Elapid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007124:Immunoenzyme Techniques; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic
D018076:DNA, Complementary; D004546:Elapid Venoms; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000110:Acetylcholinesterase