Isolation of cDNAs encoding cellular drug-binding proteins using a novel expression cloning procedure: drug-western.

Abstract

A rapid and convenient new method for isolating the genes encoding cellular drug-binding proteins is described. This method, drug-western, is based on the use of the drug conjugated with a marker molecule as a probe for the screening of a cDNA library. Unlike the other methods, this method allows us to identify the genes for trace amounts of cellular drug-binding proteins without purification. We have used this approach to isolate human cDNA clones encoding binding proteins of HMN-154 ((E)-4-[2-[2-(p-methoxy-benzene-sulfonamide) phenyl]ethenyl] pyridine), a novel benzenesulfonamide anticancer compound (Katoh and Hidaka 1997). The proteins encoded by two of the isolated clones are identical to NF-YB, B subunit of nuclear transcription factor NF-Y, and thymosin beta-10, respectively. Recombinants of both proteins bind specifically to HMN-154 in vitro. Comparison of amino acid sequences between these proteins shows the sequence similarity in a short amino acid stretch [K(X)AKXXK]. Deletion or mutation of this region causes the significant loss of binding of both proteins to HMN-154. Furthermore, HMN-154 inhibits DNA binding of NF-Y to the human major histocompatibility complex class II human leukocyte antigen DRA Y-box sequence in a dose-dependent manner. Interestingly, other binding proteins identified by this method also possess the same or a similar motif. These results clearly demonstrate that NF-YB and thymosin beta-10 are specific cellular binding proteins of HMN-154 and that this shared region is necessary for the binding to HMN-154. Hence, this new method is thought to be useful for the identification of drug-binding proteins.