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DNA Research 2005 12(4):257-267; doi:10.1093/dnares/dsi010
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© The Author 2006. 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

Preparation of a Set of Expression-Ready Clones of Mammalian Long cDNAs Encoding Large Proteins by the ORF Trap Cloning Method

Daisuke Nakajima1, Kenji Saito2, Hisashi Yamakawa1, Reiko F. Kikuno1, Manabu Nakayama1,2, Reiko Ohara1, Noriko Okazaki1, Hisashi Koga1,3, Takahiro Nagase1,* and Osamu Ohara1,2,4

1Department of Human Gene Research, Kazusa DNA Research Institute 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
2Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
3Chiba Industry Advancement Center 2-6 Nakase, Mihama-ku, Chiba 261-7126, Japan
4RIKEN Research Center for Allergy and Immunology 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS–polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.

Key words: large protein; cDNA; expression clone; proteomics; subcellular localization

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

Communicated by Michio Oishi


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T. Nagase, H. Yamakawa, S. Tadokoro, D. Nakajima, S. Inoue, K. Yamaguchi, Y. Itokawa, R. F. Kikuno, H. Koga, and O. Ohara
Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products
DNA Res, June 1, 2008; 15(3): 137 - 149.
[Abstract] [Full Text] [PDF]


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