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Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14652-7. Epub 2002 Oct 30.

A cell-free protein synthesis system for high-throughput proteomics.

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  • 1Department of Applied Chemistry, Faculty of Engineering, and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.

Abstract

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

PMID:
12409616
[PubMed - indexed for MEDLINE]
PMCID:
PMC137474
Free PMC Article

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