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A wheat germ cell‐free system is a novel way to screen protein folding and function
Author(s) -
Morita Eugene Hayato,
Sawasaki Tatsuya,
Tanaka Rikou,
Endo Yaeta,
Kohno Toshiyuki
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0241203
Subject(s) - cell free protein synthesis , wheat germ , cell free system , heteronuclear single quantum coherence spectroscopy , folding (dsp implementation) , protein biosynthesis , protein folding , function (biology) , biochemistry , escherichia coli , chemistry , macromolecule , biology , cell , microbiology and biotechnology , nuclear magnetic resonance spectroscopy , enzyme , gene , stereochemistry , engineering , electrical engineering
Abstract For high‐throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell‐free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15 N‐labeled proteins and obtained 1 H‐ 15 N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and 1 H‐ 15 N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell‐free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell‐free protein synthesis system to high‐throughput protein structural analysis.