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

FEBS Letters

Depicker, Ann¹  Fiers, Esbjörn¹  De Jaeger, Geert¹  Eeckhout, Dominique¹ 

[1] Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

关键词: Dihydroflavonol-4-reductase;    Two-hybrid;    Immunomodulation;    Reducing subcellular environment;    Single-chain variable fragment;    DFR;    dihydroflavonol-4-reductase;    OD;    optical density;    scFv;    single-chain variable fragment;    TSP;    total soluble protein;   

DOI  :  10.1016/S0014-5793(00)01182-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown