The improved method for identification of drug-target molecules under yeast inducible expression system.
Kaoru Ogawa, Makiyo Hinata, Toru Arai, Kappei Tsukahara, Junro Kuromitsu, Takeshi Nagasu
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, 300-2635, Japan
Multicopy suppressor screening is one of the methods for identification of drug-target molecules because overexpression of the protein often overcomes the drug effect such as growth inhibition. However there are many compounds that have no effect on yeast growth due to its low permeability. Disruption of genes required for expression of drug-efflux pumps increase drug sensitivity. In addition, deletion of ERG6 gene additively allows cells to become more sensitive for many drugs. In spite of increasing drug sensitivity, transformation efficiency was significantly reduced in the erg6 mutant strain. To solve this problem, we have made the strain that could induce the expression of ERG6 under GAL10 promoter. This ERG6 -inducible strain showed the same drug sensitivity as the erg6 deletion strain in the glucose-containing medium and could be transformed with high efficiency in galactose-containing medium. To assess the feasibility of the strain, we have introduced the HeLa cDNA library with high complexity and obtained sufficient number of transformants of the ERG6 -inducible strain. Moreover, the strain was applied for the multicopy suppressor screening of anticancer drug Methotrexate (MTX), resulted in successfully identifying the target molecule dihydrofolate reductase. These results indicate that drug-target molecules in the human cells could be identified in this ERG6 -inducible expression system.