Cell-free protein synthesis and

Protein synthesis in extracts of cells infected with other Ml viruses is not inhibited to the same extent as in the case of picornaviiTuses. This has been clearly shown for VSV extracts prepared from HeLa cells at different times after infection with VSV are very active in cell-free protein synthesis and synthesize only viral proteins (our unpublished observations). The replicative intermediate of VSV does not seem capable of activating the protein kinase of HeLa cells and of inhibiting protein synthesis. 

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. 

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. 

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). 

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.

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. 

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. 

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.

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.

In order to obtain large amount of proteins with this wheat germ cell-free protein synthesis system, even trace contaminations of ribonucleases should be avoided throughout the transcription and translation steps. Therefore, great care must be... 

While the complementary double helical structure explained how particular sequences of bases could be used to store a genetic instruction it was not immediately clear how replication occurred or, indeed, how these instructions were used. Later work by Gamow linked DNA base pair sequences to protein synthesis [15] but it was not until 1961, when Nirenberg and Matthaei demonstrated that cell-free protein synthesis relied upon synthetic or natural polynucleotides [16], that the final link was made. The information held within the linear DNA sequence is replicated every time a cell divides. Replication is possible because of the unique double helical structure of DNA as shown in Fig. 2.7. 

J. H. Matthaei, 0. W. Jones, R. G. Martin, and S. H. Barondes Approximation of genetic code via cell-free protein synthesis directed by template RNA. Federation Proc. 22, 55 (1963). 

Kim, D. M. and J. R. Swartz. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol Bioeng (1999) 66(3) 180-8. 

Another approach to inexpensive labelling of proteins is by means of cell-free protein synthesis.48 50 These in vitro expression systems use a crude Escherichia coli fraction as a source of ribosomes and other factors necessary for the synthesis of proteins, T7 RNA polymerase for transcription, and dialysis to maintain levels of amino acids and nucleotides. In principle, the technology is capable of labelling proteins at specific sites without isotope scrambling and also of producing labelled proteins from small volumes of labelled medium. 

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