in vitro Protein Synthesis Kit PURESYSTEM® Kit flyer download [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 .
Studies using PURESYSTEM
- Shimizu Y, Ueda T., SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation., J Biol Chem. 2006 Jun 9;281(23):15987-96. Epub 2006 Apr 6.
- Kaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA, Hayer-Hartl M, Hartl FU, Barral JM. Real-time observation of trigger factor function on translating ribosomes. Nature. 2006 Nov 23;444(7118):455-60. Epub 2006 Oct 15.
- Tomic S, Johnson AE, Hartl FU, Etchells SA. Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains. FEBS Lett. 2006 Jan 9;580(1):72-6. Epub 2005 Dec 6.
- Kuruma Y, Nishiyama K, Shimizu Y, Muller M, Ueda T. Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol Prog. 2005 Jul-Aug;21(4):1243-51.
- Shimizu Y, Kanamori T, Ueda T. Protein synthesis by pure translation systems. Methods. 2005 Jul;36(3):299-304.
- Itoh H, Kawazoe Y, Shiba T. Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system. J Microbiol Methods. 2005 Jun 23;
- Sando S, Kanatani K, Sato N, Matsumoto H, Hohsaka T, Aoyama Y. A small-molecule-based approach to sense codon-templated natural-unnatural hybrid peptides. Selective silencing and reassignment of the sense codon by orthogonal reacylation stalling at the single-codon level. J Am Chem Soc. 2005 Jun 8;127(22):7998-9.
- Fukushima K, Ikehara Y, Yamashita K. Functional role played by the glycosylphosphatidylinositol anchor glycan of CD48 in interleukin-18-induced interferon-gamma production. J Biol Chem. 2005 May 6;280(18):18056-62. Epub 2005 Mar 10.
- Yano M, Okano HJ, Okano H. Involvement of Hu and heterogeneous nuclear ribonucleoprotein K in neuronal differentiation through p21 mRNA post-transcriptional regulation. J Biol Chem. 2005 Apr 1;280(13):12690-9.
- Ying BW, Taguchi H, Kondo M, Ueda T. Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides. J Biol Chem. 2005 Mar 25;280(12):12035-40.
- Tanaka R, Mizukami M, Tokunaga M. Novel processing and localization of catA, ccdA associated thiol-disulfide oxidoreductase, in protein hyper-producing bacterium Brevibacillus choshinensis. Protein Pept Lett. 2005 Jan;12(1):95-8.
- Ying BW, Taguchi H, Ueda H, Ueda T. Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system. Biochem Biophys Res Commun. 2004 Aug 6;320(4):1359-64.
- Asai T, Takahashi T, Esaki M, Nishikawa S, Ohtsuka K, Nakai M, Endo T. Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import. J Biol Chem. 2004 May 7;279(19):19464-70.
- Kawano M, Suzuki S, Suzuki M, Oki J, Imamura T. Bulge- and basal layer-specific expression of fibroblast growth factor-13 (FHF-2) in mouse skin. J Invest Dermatol. 2004 May;122(5):1084-90.
- Udagawa T, Shimizu Y, Ueda T. Evidence for the translation initiation of leaderless mRNAs by the intact 70S ribosome without its dissociation into subunits in eubacteria. J Biol Chem. 2004 Mar 5;279(10):8539-46.
- Ying BW, Suzuki T, Shimizu Y, Ueda T. A novel screening system for self-mRNA targeting proteins. J Biochem (Tokyo). 2003 Apr;133(4):485-91.
- Shimizu Y, Ueda T. The role of SmpB protein in trans-translation. FEBS Lett. 2002 Mar 6;514(1):74-7.