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, G 4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybri da, and the DFR-encoding sequence were cloned in two-hybrid vectors. The ve ctors 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 me dium without histidine. ScFv-G4 and, to a lesser extent, scFv-A4 could inte ract with DFR in the east nucleus. On the contrary, scFv-H3 showed no inter action with its antigen in yeast. The results of a previous expression anal ysis of the same scFv Fragments in the plant cytosol correlate with those o f the two-hybrid test. This suggests that it is possible to evaluate the an tigen-scFv interaction in a reducing subcellular environment with the two-h ybrid test. Therefore, the yeast two-hybrid system tan be useful to identif y candidate scFv fragments for intracellular antibody applications. (C) 200 0 Federation of European Biochemical Societies.