Identification of the anti-protozoal compound binding protein for the development and application of novel drug targets.

In the process of searching for antimalarial drugs, it was found that nilotinib, a Bcr-Abl tyrosine kinase inhibitor used as a treatment for chronic myelogenous leukemia, exhibits antimalarial activity (J. Antibiot., 2014). On the other hand, since malaria parasites do not have tyrosine kinases, nilotinib is expected to exhibit antimalarial activity through a new mechanism of action. By clarifying the antimalarial action target, the research is being conducted with the aim of developing new drug discovery targets and applying them to screening for drug discovery. A nilotinib-binding protein, pfeIF4A, was found by target protein fishing using the cytoplasmic fraction of the malaria parasite. His-tagged pfeIF4A could not be obtained until verification, and the amount of production was small. In addition, since the expression of His-tagged pfeIF4A was not reproduced, various E. coli strains were used, and the expression of the protein was reproduced by using E. coli BL21(DE3)[pLysS] obtained from Stratagene. Culture conditions The cultured cells examined are stored at -80°C, and the expression is confirmed and purified one by one, and the protein is secured while unifying the expression conditions. In addition to the E. coli expression system, the study investigated the acquisition of His-tagged pfeIF4A by a cell-free protein expression system for the purpose of more efficient protein expression and acquisition. His-tagged detection of the post-translational reaction mixture was positive, but SDS-PAGE showed that the size was different from the intended size. It is also possible that the translation factor in the reaction system and the expressed His-tagged pfeIF4A were associated as a translation factor complex and detected at different sizes, which needs to be confirmed by Western blotting.

Project details

Basic Science

Apr 2020 — Mar 2023

$38,109

Japan