A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era. ß

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.

We report a morphological study of functioning ribosomes in a e⁄cient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having di¡erent 5P P and 3P P untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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