The 30th International Conference on Arabidopsis Research (ICAR 2019)

697 / 2019-05-01 16:38:09

Production and characterization of polyclonal antibody against Arabidopsis TOR and its cross-activity

Polyclonal antibody,Truncation,Arabidopsis thaliana,Target of rapamycin (TOR)

Plenary track > Metabolism

TARGET OF RAPAMYCIN (TOR) kinase is encoded by large gene and a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. TOR plays a master regulator that integrates nutrients, energy and stress signaling to synchronize developmental processes of cells. TOR was first identified in budding yeast via genetic mutant screening by the resistance to rapamycin which has been well-studied in animals and yeast due to its potent immunosuppressive and cytostatic anti-cancer activity. However, little is known about TOR in plants due to the lack of reliable molecular and biochemical assay tools to detect TOR protein activities. Here, we produced polyclonal α-TOR antibody using truncated variants of TOR having amino-terminal (1-200 aa and 1113-1304aa) regions as antigens. Both recombinant His-TOR1-200 and His-TOR 1113-1304 proteins were individually expressed in E. coli and its protein mixtures (as a concentration ratio of 1:1) were immunized into rabbit. Anti-serum was purified by antigen-specific affinity purification method using both recombinant His-TOR1-200 and His-TOR 1113-1304 proteins. Purified polyclonal α-TOR antibody successfully detected not only endogenous TOR proteins in wild-type Arabidopsis plants, but also its ortholog proteins in crop plants including tomato, corn and alfalfa. Our polyclonal Arabidopsis TOR antibody could help to determine the precise molecular mechanisms of TOR which has largely unknown pleiotropic responses in plants.