Proteins cell-free synthesis

Enhancement of the catalytic activity of the wheat germ, cell-free, protein synthesis system using a fortified translation extract... 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

Sawasaki, T., Ogasawara, T., Morishita, R. and Endo, Y. (2002) A cell-free protein synthesis system for high-throughput proteomics. Proceedings of the National Academy of Sciences of the United States of America, 99 (23), 14652-14657. 

Jackson, A.M., Boutell, J., Cooley, N. and He, M. (2004) Cell-free protein synthesis for proteomics. Briefings in Functional Genomics and Proteomics, 2 (4), 308-319. 

Morita, E.H., Shimizu, M., Ogasawara, T. et al. (2004) A novel way of amino acid-specific assignment in1 II-15N HSQC spectra with a wheat germ cell-free protein synthesis system. Journal of Biomolecular NMR, 30 (1) 37 15. 

Montesano, L., Cawley, D., and Herschman, H.R. (1982) Disuccinimidyl suberate cross-linked ricin does not inhibit cell-free protein synthesis. Biochem. Biophys. Res. Comm. 109, 7-13. 

Asano, K., B. Svensson, and F. M. Polsen. Isolation and characterization of inhibitors of animal cell-free protein synthesis from barley seeds. Carlsberg Res Commun 1984 49(7) 619—626. Roberts, W. K., and C. P. Selitren-nikoff. Isolation and partial characterization of two antifungal proteins from barley. Biochim Biophys Acta 1986 880 161-170. 

The Transdirect insect cell is a newly developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Spodoptera fru iperda 21 (S 21) insect cells. An expression vector, pTDl, which includes a 5 -imtranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of about 50 pg per mL of the translation reaction mixture. This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts. 

Key words Translation, Spodoptemfrupiperdall, Cell-free protein synthesis system, pTDl vector. Insect cell extract... 

In reverse chemical genetics, it is crucial to synthesize proteins of interest using appropriate foreign gene expression systems and cDNA resources. Since cell-free protein synthesis systems have the potential to synthesize any desired proteins, including both native proteins and those that are toxic to cells (1), with high throughput, they can be powerful tools for this objective. We developed a cell-free protein synthesis system from Spodop-tera fm iperda 21 (S 21) insect cells, which are widely used as the host for baculovirus expression systems, and commercialized it as the Transdirect insect cell. 

We have demonstrated that this insect cell-free protein synthesis system is one of the most effective protein synthesis systems among those based on animal extracts (2). Furthermore, it has the potential to perform eukaryote-specific protein modifications such as protein W-myristoylation and prenylation (3, 4). Thus, we expect that the insect cell-free protein synthesis system will be a useful method for target protein production in the reverse chemical genetics era, as well as for postgenomic studies. In this chapter, we describe standard protocols to synthesize proteins of interest using the insect cell-free protein synthesis system. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Construction of the Expression Clone for the Insect Cell-Free Protein Synthesis System... 

Fig. 3. Detection of a synthesized protein by fluorescent labeling. Cell-free protein synthesis was carried out with or without the use of mRNA transcribed from a linearized expression done containing the p-gaiactosidase gene, and the synthesized protein was labeled by FluoroTect. The translational reaction mixtures were resolved by 12.5% SDS-PAGE. Detection of labeled protein was performed using a laser-based fluorescent scanner (FX pro, Bio-Rad, Hercules, CA). Lanes 1 and 2 represent negative control (absence of mRNA) and p-galactosidase, respectively.

The pTDl vector should not be used as the expression vector for an E. coli cell-free protein synthesis system because this vector does not contain Shine-Dalgarno sequence. 

Sakurai, N., Moriya, K., Suzuki, T., Sofiiku, K, Motiki, H., Nishimura, O., and Utsmni, T. (2007) Detection of co- and post-translational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system. Anal. Biochem. 362, 236-244. 

Ezure, T., Suzuki, T., Higashide, S., Shintani, E., Endo, K., Kobayashi, S., Shikata, M., Ito, M., Tanimizu, K., and Nishimiu-a, O. (2006) Cell-free protein synthesis system prepared from insect cells by freeze-thawing. Biotechnol. Pro. 22, 1570-1577. 

Liposomes from EggPC, cholesterol, DSPE-PEG5000 used to entrap cell-free protein synthesis. 

Cell-free protein synthesis through solubilisate exchange in water/oil emulsion compartments. Chem. Bio. Chem, 5, 1055-1062. 

Spirin, A.S., and Swartz, J.R. (2008) Cell-free protein synthesis systems historical landmarks, classification, and general methods, in Cell-Free Protein Synthesis (eds A.S. Spirin and J. R. Swartz), Wiley-VCH Verlag GmbH, Weinheim, Germany, pp. 1-34. 

Voloshin, A.M., and Swartz, J.R. (2008) Large-scale batch reactions for cell-free protein synthesis, in Cell-Free Protein... 

Nirenberg, M. W., and J. H. Mattaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. 

Key Words Cell-free protein synthesis pure embryo isolation wheat extract preparation transcription and translation reactions. 

CFCF, continuous-flow cell-free protein synthesis system PCR, polymerase chain reaction TB, transcription buffer. 

Fig. 3. (Opposite page) A novel bilayer cell-free protein synthesis. (A) Schematic illustration of the method. Wheat embryo cell-free system as described under Subheading 4.2. (B) Synthesis of green fluorescent protein (GFP) by bilayer mode. (C) The bilayer method ( ), bilayer but mixed ( ). For the measurement of (14C)leucine incorporation, samples were vortexed and hot trichloroacetic acid-insoluble radioactivity in 5 pL in the batch reaction or 30 pL in the bilayer reaction, thus adjusted amount of extracts in each system. The inset shows autoradiograms, and arrowheads mark GFP. Reprinted from (16) by permission of Federation of the European Biochemical Societies.

The wheat germ cell-free protein synthesis system has been widely used to express various kinds of proteins. The activity of this system, however, was very low. Madin et al. found that this low activity was due to contamination by... 

Fig. 5. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of 15N-labeled yeast ubiquitin synthesized by wheat germ cell-free protein synthesis system. Left lane molecular weight markers. Middle lane yeast ubiquitin translation solution. Right lane wheat germ alone (control). Yeast ubiquitin is indicated by...

Fig. 7. H-15N heteronuclear single-quantum coherence (HSQC) spectrum of yeast ubiquitin synthesized by wheat germ cell-free protein synthesis system, not purified (0.1 vaM, 128 [tl] 512 [t2] complex points, 512 scans), obtained at the H resonance frequency of 500 MHz. Spectral widths are 1600 and 6250 Hz in FI and F2, respectively.

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