The wheat germ cell-free system is composed out of five basic steps, growth of Escherichia coli harboring plasmid, first colony-PCR, second PCR, transcription, and translation. Improvements of culture medium, colony based PCR, and modifications within the split primer set of the second PCR amplify both DNA and RNA levels. This yields more than 5 times increase in protein amount for pEU-originated templates. Especially, for the low PCR-amplifiable vectors with pET-origin, it leads to 30 fold higher product amount in translation. This broadens the range of usable vectors, overcoming the existing cell-free system limitations for high-throughput protein screening. Noteworthily, the system successfully maintains translation by S-30 cell-free extract below 30 OD. In conclusion, this improved firm cell-free system reduces cost and enables robotic automation and high-throughput thermodynamic analysis, especially for proteins that are difficult to be expressed. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.