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Journal of Biochemistry Advance Access published online on August 17, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj174
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© 2006 The Japanese Biochemical Society.
Received May 21, 2006
Accepted August 4, 2006

Regular Paper

A Novel Way to Express Proline-Selectively Labeled Proteins with a Wheat Germ Cell-Free Protein Synthesis System

Masato Shimizu 1, Takahisa Ikegami 2, Koichi Akiyama 1, and Eugene Hayato Morita 3 *

1 Division of Gene Research, Department of Molecular Science, Integrated Center for Science, Ehime University, 3-5-7 Tarumi, Ehime 790-8566, Japan
2 Laboratory of Structural Proteomics, Research Center for Structural and Functional Proteomics, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565-0871, Japan
3 Division of Gene Research, Department of Molecular Science, Integrated Center for Science, Ehime University, 3-5-7 Tarumi, Ehime 790-8566, Japan; Satellite Venture Business Laboratory, Ehime University, 3-5 Matsuyama, Ehime 790-8577, Japan

* To whom correspondence should be addressed.
Eugene Hayato Morita, E-mail: ehmorita{at}dpc.ehime-u.ac.jp


  Abstract

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.

Keywords: cell-free; HNCO; proline; selective labeling; wheat germ.
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