A wheat germ cell-free system is a novel way to screen protein folding and function.
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.
Morita
Eugene Hayato
EH
Center for Gene Research, Ehime University, Ehime 790-8566, Japan. ehmorita@dpc.ehime-u.ac.jp
Sawasaki
Tatsuya
T
Tanaka
Rikou
R
Endo
Yaeta
Y
Kohno
Toshiyuki
T
D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D017510:Protein Folding; D040901:Proteomics; D013329:Structure-Activity Relationship; D014908:Triticum; D025801:Ubiquitin / Q000096:biosynthesis / Q000737:chemistry
D010940:Plant Proteins; D025801:Ubiquitin