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J Struct Funct Genomics. 2004;5(1-2):45-57.

High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.

Author information

  • 1Cell-Free Science and Technology Research Center, and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan. yendo@en3.ehime-u.ac.jp

Abstract

Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.

PMID:
15263842
[PubMed - indexed for MEDLINE]
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