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

Hideki Tanaka, Nobuko Ohshima, Hiroyoshi Hidaka

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 β-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 β-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.

Original languageEnglish
Pages (from-to)356-363
Number of pages8
JournalMolecular Pharmacology
Volume55
Issue number2
DOIs
Publication statusPublished - 01-01-1999

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Organism Cloning
Carrier Proteins
Complementary DNA
Pharmaceutical Preparations
Thymosin
Clone Cells
HLA-D Antigens
Sequence Deletion
HLA Antigens
HMN 154
Major Histocompatibility Complex
Gene Library
Recombinant Proteins
Genes
Amino Acid Sequence
Proteins
Transcription Factors
Amino Acids
DNA

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

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Isolation of cDNAs encoding cellular drug-binding proteins using a novel expression cloning procedure : Drug-western. / Tanaka, Hideki; Ohshima, Nobuko; Hidaka, Hiroyoshi.

In: Molecular Pharmacology, Vol. 55, No. 2, 01.01.1999, p. 356-363.

Research output: Contribution to journalArticle

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