Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system.
The coding sequences of three single-chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybrida, and the DFR-encoding sequence were cloned in two-hybrid vectors. The vectors were transformed in the yeast strain HF7c (his3-200, trp1-901, leu2-3) and the scFv-DFR interaction was analyzed by measuring yeast growth on medium without histidine. ScFv-G4 and, to a lesser extent, scFv-A4 could interact with DFR in the yeast nucleus. On the contrary, scFv-H3 showed no interaction with its antigen in yeast. The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two-hybrid test. This suggests that it is possible to evaluate the antigen-scFv interaction in a reducing subcellular environment with the two-hybrid test. Therefore, the yeast two-hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.
De Jaeger
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Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Fiers
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Eeckhout
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Depicker
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D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000946:Antigens, Fungal / Q000276:immunology*; D003001:Cloning, Molecular; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007135:Immunoglobulin Variable Region; D010084:Oxidation-Reduction; D010944:Plants; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D000946:Antigens, Fungal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase