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DNA Research Advance Access published online on March 2, 2008
DNA Research, doi:10.1093/dnares/dsn004
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© The Author 2008. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Takahiro Nagase1,*, Hisashi Yamakawa1, Shinichi Tadokoro2, Daisuke Nakajima1, Shinichi Inoue1, Kei Yamaguchi3, Yasuhide Itokawa4, Reiko F. Kikuno1, Hisashi Koga1 and Osamu Ohara1,2,5

1 Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
2 Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
3 Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan
4 Graduate School of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
5 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

Received 10 January 2008 ; accepted 29 January 2008.

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi® cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag® labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi® ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.

Key words: cDNA; HaloTag; proteomics; Flexi cloning; ORFeome

* To whom correspondence should be addressed. Tel. +81 438-52-3930. Fax. +81 438-52-3931. E-mail: nagase{at}kazusa.or.jp

Edited by Katsumi Isono


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