Resource abstract
Nature Methods 5, 1011 - 1017 (2008)
Published online: 23 November 2008 | doi:10.1038/nmeth.1273
Human protein factory for converting the transcriptome into an in vitro–expressed proteome
Naoki Goshima1, Yoshifumi Kawamura1,2, Akiko Fukumoto1,2, Aya Miura1,2, Reiko Honma3, Ryohei Satoh2, Ai Wakamatsu1,4,5, Jun-ichi Yamamoto5, Kouichi Kimura6, Tetsuo Nishikawa5,6, Taichi Andoh2, Yuki Iida7, Kumiko Ishikawa2, Emi Ito3, Naoko Kagawa2, Chie Kaminaga2, Kei-ichi Kanehori7, Bunsei Kawakami8, Kiyokazu Kenmochi2, Rie Kimura2, Miki Kobayashi9, Toshihiro Kuroita8, Hisashi Kuwayama2, Yukio Maruyama2, Kiyoshi Matsuo7, Kazuyoshi Minami2, Mariko Mitsubori2, Masatoshi Mori1,2, Riyo Morishita1, Atsushi Murase2, Akira Nishikawa3, Shigemichi Nishikawa10, Toshihiko Okamoto2, Noriko Sakagami2, Yutaka Sakamoto2, Yukari Sasaki2, Tomoe Seki1,2, Saki Sono2, Akio Sugiyama2, Tsuyoshi Sumiya2, Tomoko Takayama1,2, Yukiko Takayama7, Hiroyuki Takeda2, Takushi Togashi1,2, Kazuhide Yahata2, Hiroko Yamada2, Yuka Yanagisawa3, Yaeta Endo11, Fumio Imamoto12, Yasutomo Kisu1,2, Shigeo Tanaka2, Takao Isogai4,5, Jun-ichi Imai3, Shinya Watanabe3 & Nobuo Nomura1
Abstract
Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro–synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
- National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan.
- Japan Biological Informatics Consortium, TIME24 bldg. 10F, 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan.
- Department of Clinical Informatics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
- Reverse Proteomics Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0818, Japan.
- Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., 1-280 Higashi-koigakubo, Kokubunji, Tokyo 185-8601, Japan.
- Hitachi Science Systems, Ltd., 1040 Ichige, Hitachinaka, Ibaraki 312-0033, Japan.
- Toyobo Co., Ltd., Tsuruga Institute of Biotechnology, 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan.
- Mitsubishi Chemical Group, Science and Technology Research Center Inc., 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan.
- Wakenyaku Co., Ltd., Research and Development Div.–Kusatsu Center, 945-1 Tsujide Shimogasa-cho, Kusatsu, Shiga 525-0029, Japan.
- Department of Applied Chemistry, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Correspondence to: Naoki Goshima1 e-mail: n-goshima@aist.go.jp
Correspondence to: Nobuo Nomura1 e-mail: nomura.88@aist.go.jp
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