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Practical cell-free protein synthesis system using purified wheat embryos.

Nature protocols (2010-02-06)
Kazuyuki Takai, Tatsuya Sawasaki, Yaeta Endo
ABSTRACT

Biochemical characterization of each gene product encoded in the genome is essential to understand how cells are regulated. The bottleneck has been and still is in how the gene products can be obtained. The wheat cell-free protein synthesis system we have developed is a powerful method for preparation of many different proteins at a time and also for preparation of large amounts of specific proteins for biochemical and structural analyses. Here, we show a method for preparation of the wheat embryo extract useful for the cell-free reactions, by which 5 ml of a high-activity extract is obtained in 4-5 d. We also describe the methods for small- and large-scale protein synthesis by hands-down operations with the use of mRNAs prepared by transcription of PCR products and pEU plasmids harboring the target cDNAs, which need 2-4 d excepting the time required for plasmid preparation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Cytidine 5′-triphosphate disodium salt, ≥95%
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, Grade II, ≥97% (HPLC), crystalline, from microbial
Sigma-Aldrich
Uridine 5′-triphosphate trisodium salt hydrate, from yeast, Type III, ≥96%