Cell-free products for NMR measurements do not need extensive purification, because none of the endogenous proteins are labeled during the translation. Cell-free systems derived from E. coli, however, contain high levels of amino-acid-metabolizing enzymes, since the extracts are prepared from cells in the exponential growth phase. As a result, many types of inhibitors of amino acid metabolism or even suitable mutant strains have to be added to avoid scrambling of the label among amino acids. In the wheat-based system, in contrast, most of those enzyme activities were expected very low, since the embryos are in hibernation. Nevertheless, we found that two transaminases and one synthetase were active during the translation reaction. Our subsequent search for their inhibitors were successful in that the interconversions of amino-15N between Ala and Glu (alanine transaminase) and between Glu and Asp (asparatate transaminase) were completely inhibited by adding P-chloro-L-alanine and L-methionine sulfoximimate, and that the other leaking pathway between Glu and Asp (glutamine synthetase) was suppressed by aminooxyacetate [22]. Incorporation of amino acids into protein is not very high even in our system. This economical issue could be solved by recycling the amino acids left in the mixture using the conventional method for purifying amino acids after translation. This is another attractive feature that benefits the NMR field. The improved methodology may pave the way for a high-throughput protein synthesis system suited for estimating fold-edness [23, 24] and intermolecular interaction between ligands and the proteins of inters as well as assigning signals in the NMR field. A fruitful result of the application of the wheat cell-free protein synthesis system to NMR structure determination is now presented at (http://www.uwstructuralgenomics.org/) [24, 25]. The results of X-ray analyses including selenomethionine labeling for MAD phasing are to be published.

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