Reconstituted Cell-free Protein Synthesis System, PURESYSTEM - Background

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Reconstituted Cell-free Protein Synthesis System　<PURESYSTEM®> Flyer PDF

PURESYSTEM originally developed by a group of Prof. T. Ueda of the University of Tokyo is a proprietary protein synthesis and purification technology of Post Genome Institute Co., Ltd (PGI). PURESYSTEM is a novel reconstituted cell-free protein synthesis system which consists of purified components necessary for transcription, translation and energy regeneration. In PURESYSTEM, all protein factors for transcription and translation are tagged with hexahistidine: 3 initiation factors (IF1, IF2, IF3), 3 elongation factors (EF-G, EF-Tu, EF-Ts), 3 release factors (RF1, RF2, RF3), ribosome recycling factor, 20 aminoacyl-tRNA synthetases, methionyl-tRNA transformylase and T7 RNA polymerase. The reagents also contain E. coli ribosome, amino acids, NTPs, E. coli tRNA and the energy regeneration system (Figure).

Since most protein synthesis systems occur under reducing conditions, the original PURESYSTEM was also developed for a reaction under a reducing condition. However, some proteins including secretory proteins require the formation of correct disulfide bonds to achieve their activities. Synthesis of active forms of those proteins requires oxidative conditions. Taking advantages of the ability to easily change components in PURESYSTEM, the reaction condition has been adjusted from a reduced to oxidized condition by adding oxidized glutathione instead of dithiothreitol. Furthermore, addition of enzyme such as protein disulfide isomerase can aid formation of the correct disulfide bonds. ThePURESYSTEM kit suitable for synthesizing proteins with disulfide bonds is commercially available as a PURESYSTEM S-S kit from PGI. Alkaline phosphatase (AP), protein containing two disulfide bonds, was tried to be synthesized with a PURESYSTEM S-S and original PURESYSTEM systems, and the specific activities of synthesized AP proteins were compared. Although AP synthesized with the original PURE system had low activity, AP derived from the PURESYSTEM S-S kit had several-fold higher activity.

PURESYSTEM has an advantage that synthesized protein can be quickly purified using metal affinity resins and ultrafiltration membranes. Taking such an advantage, the device of PA-1 which can synthesize and purify protein with PURESYSTEM was developed .

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