Analysis of the interaction between single‐chain variable fragments and their antigen in a reducing intracellular environment using the two‐hybrid system

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Abstract
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.