Cell-free protein synthesis system

Progress has been made in demonstrating synthesis of specific proteins by cell-free systems, e.g., on the synthesis of cytochrome c by isolated rat liver mitochondria (S3, cf. 383) and on the synthesis of serum albumin by the isolated microsome fraction of rat liver 34, cf. 335,336). Campbell et al. 34) concluded that while specific serum albumin is, indeed, synthesized on the ribonucleoprotein particle fraction of the microsomes, it is not readily released in soluble form. In other words, the isolated microsomes have lost their ability to promote substrate turnover. The same is true for the hemoglobin-synthesizing RNP particles from rabbit reticulocytes 35, cf. 138). Ogata and associates 36) have essentially confirmed the results of Campbell et al. 34) on the synthesis of serum albumin by liver microsomes they have also studied the relative effect of both stimulatory and inhibitory factors on the incorporation of different amino acids into total ribonucleoprotein and into serum albumin, and showed that the requirements for the two processes were generally the same it may be noted, however, that pretreatment of the pH 5 enzymes with ribonuclease, which caused a 95% inhibition of the ineorporation into ribonucleoprotein, inhibited the corresponding incorporation into serum albumin by only 55%. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

Spirin, A.S. High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22 538-545, 2004. 

Girbes, L. Citores, and F. Soriano. Primary structure of omega-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibi- HV028 tion of protein synthesis in eucaryotic and procaryotic cell-free system. Eur J Biochem 1996 239(1) 67-73. 

Kigawa, T., Matsuda, T., Yabuki, T., and Yokoyama, S. (2008) Bacterial cell-free system for highly efficient protein synthesis, in Cell-Free Protein Synthesis (eds A.S. Spirin and J. R. Swartz), Wiley-VCH Verlag GmbH, Weinheim, Germany, pp. 83-97. 

Zamecnik and his colleagues developed the first cell-free systems for the study of protein synthesis. 

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. 9. Procedure for protein synthesis using the wheat embryo cell-free system. WEX is an abbreviation for wheat expression system.

Schachtschabel, D. and Zillig, W. (1959) Investigations on the biosynthesis of proteins. I. Synthesis of radiocarbon labeled amino acids in proteins of cell-free nucleoprotein-enzyme-system of Escherichia coli. Hoppe-Seyler s Z. Physiol. Chem. 314, 262-275. 

Kawasaki, T., Gouda, M. D., Sawasaki, T., Takai, K., andEndo, Y. (2003) Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. Eur. J. Biochem. 270, 4780-4786. 

The methods described below outline (1) the preparation of 15N-labeled protein with E. coli cells, (2) the construction of the expression plasmid for the wheat germ cell-free system, (3) the preparation of mRNA for the wheat germ system, (4) protein synthesis by the wheat germ system, (5) sample preparation for NMR, and (6) NMR analysis of the proteins. 

Protein Synthesis by the Wheat Germ Cell-Free System... 

Protein synthesis procedures using the wheat germ cell-free system are described as follows, including (a) the description of the wheat germ cell-lfee protein synthesis system developed by Madin et al., (b) the preparation of solutions for protein synthesis, and (c) the analysis of synthesized proteins. 

Cell-free systems capable of synthesising polypeptides have been prepared from protoscoleces of E. granulosus (7), larval T. crassiceps (588) and H. diminuta (633). In general, these studies have demonstrated that protein synthesis in cestodes, although showing some specificity, is similar to that in mammals in that it requires polysomes, amino acid adenylates, aminoacyl-tRNAs, pH 5 fraction, ATP, GTP, magnesium and either sodium or potassium ions. 

Parker, R. D., Jr Maclnnis, A. J. (1977). Hymenolepis diminuta isolation, purification, and reconstruction in vitro of a cell-free system for protein synthesis. Experimental Parasitology, 41 2-16. 

Ricin is extremely toxic to eukaryotic cells.32,150,194,642,844,646,647,649,657 The experiments of Olsnes and Pihl150,639,649 and Pappenheimer and coworkers658 demonstrated that one of the two subunits binds to the cell membrane, presumably by way of a carbohydrate structure, whereas the second subunit inhibits protein synthesis by a catalytic mechanism in a cell-free system. This suggests that toxicity may result from the... 

Peptide synthesis is a preparative technique as, to some extent, is protein synthesis in cell-free systems (Bodansky and Bodansky 1994 Pennington and Dunn 1994 ... 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

Information on archaeal translation is essentially based on poly(U)- and poly(UG)-programmed cell-free systems and on peptidyltransferase assay systems. Poly(U)-directed systems have been used to monitor the reconstruction of archaeal ribosomal subunits and the susceptibility of archaea to protein synthesis inhibitors. 

The elucidation of the mechanism of biosynthesis of penicillin stemmed from the discovery that isotopically labelled cysteine and valine were used in the assembly of penicillin by Penicillium chrysogenum (Amstein and Grant, 1954 Amstein and Clubb, 1957). Cysteine and valine together with a-aminoadipic acid are used by Cephalosporium acremonium to synthesise penicillin N (8.27) and cephalosporin C (8.28). Evidence was accumulated that a tripeptide, h-(f.-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) was formed as an intermediate. Since this tripeptide is not transported into mycelial cells, it must be synthesised intracellularly and synthesis of penicillin from the isotopically labelled tripeptide was demonstrated using a cell-free system. Clearly, ACV is not produced by a ribosomal synthesis of a protein followed by proteolytic processing. The enzyme involved, ACV synthetase, not only forms the two peptide bonds but also epimerises the valine residue. Thus, incubation of [2-2H]-valine with purified ACV synthetase completely removed deuterium... 

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