A wheat germ cell-free system is a novel way to screen protein folding and function

Protein Sci. 2003 Jun;12(6):1216-21. doi: 10.1110/ps.0241203.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Magnetic Resonance Spectroscopy
  • Plant Proteins / biosynthesis
  • Plant Proteins / chemistry*
  • Plant Proteins / physiology*
  • Protein Folding*
  • Proteomics
  • Structure-Activity Relationship
  • Triticum*
  • Ubiquitin / biosynthesis
  • Ubiquitin / chemistry

Substances

  • Plant Proteins
  • Ubiquitin