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A Screening of a Library of T7 Phage-Displayed Peptide Identifies E2F-4 as an Etoposide-Binding Protein
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641, Yamazaki, Noda, Chiba 278-8510, Japan
* Author to whom correspondence should be addressed.
Received: 1 March 2011; in revised form: 22 April 2011 / Accepted: 16 May 2011 / Published: 24 May 2011
Abstract: Etoposide (VP-16) is an anti-tumor compound that targets topoisomerase II (top II). In this study, we have identified an alternative binding protein of etoposide by screening a library of T7 phage-displayed peptides. After four rounds of selection using a biotinylated etoposide derivative immobilized on a streptavidin-coated plate, T7 phage particles that display a 16-mer peptide NSSASSRGNSSSNSVY (ETBP16) or a 10-mer NSLRKYSKLK (ETBP10) were enriched with the ratio of 40 or 11 out of the 69 clones, respectively. Binding of etoposide to these peptides was confirmed by surface plasmon resonance (SPR) analysis, which showed ETBP16 and ETBP10 to have a kinetic constant of 4.85 × 10−5 M or 6.45 × 10−5 M, respectively. ETBP16 displays similarity with the ser-rich domain in E2F-4, a transcription factor in cell cycle-regulated genes, suggesting that etoposide might interact with E2F-4 via this domain. SPR analysis confirmed the specific binding of etoposide to recombinant E2F-4 is in the order of 10−5 M. Furthermore, etoposide was shown to inhibit luciferase reporter gene expression mediated by the heterodimeric E2F-4/DP complex. Taken together, our results suggest that etoposide directly binds to E2F-4 and inhibits subsequent gene transcription mediated by heterodimeric E2F-4/DP complexes in the nucleus.
Keywords: etoposide (VP-16); T7 phage display; peptide; E2F-4; transcription; surface plasmon resonance
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Takami, M.; Takakusagi, Y.; Kuramochi, K.; Tsukuda, S.; Aoki, S.; Morohashi, K.; Ohta, K.; Kobayashi, S.; Sakaguchi, K.; Sugawara, F. A Screening of a Library of T7 Phage-Displayed Peptide Identifies E2F-4 as an Etoposide-Binding Protein. Molecules 2011, 16, 4278-4294.
Takami M, Takakusagi Y, Kuramochi K, Tsukuda S, Aoki S, Morohashi K, Ohta K, Kobayashi S, Sakaguchi K, Sugawara F. A Screening of a Library of T7 Phage-Displayed Peptide Identifies E2F-4 as an Etoposide-Binding Protein. Molecules. 2011; 16(5):4278-4294.
Takami, Mihoko; Takakusagi, Yoichi; Kuramochi, Kouji; Tsukuda, Senko; Aoki, Satoko; Morohashi, Kengo; Ohta, Keisuke; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio. 2011. "A Screening of a Library of T7 Phage-Displayed Peptide Identifies E2F-4 as an Etoposide-Binding Protein." Molecules 16, no. 5: 4278-4294.