Cell free synthesis

Cell free synthesis of the pectic polysaccharide homogalacturonan... 

Mohnen, D., Doong, R.L., Liljebjelke, K., FraUsh, G., and Chan, J. (1996) Cell free synthesis of the pectic polysaccharide homogalacturonan. In Pectins and Pectinases -proceedings of a conference. Anonymous Elsevier Science B.V. Amsterdam. 

Kigawa, T., Yamaguchi-Nunokawa, E., Kodama, K. et al. (2002) Selenomethionine incorporation into a protein by cell-free synthesis. Journal of Structural and Functional Genomics, 2 (1), 29-35. 

John, D.C.A., Grant, M.E. and Bulleid, N.J. (1993) Cell-free synthesis and assembly of prolyl 4-hydroxylase - the role of the beta-subunit (pdi) in preventing misfolding and aggregation of the alpha-subunit. EMBOJoumal 2,1587-1595. 

Additional information <1-7, 11, 14, 15, 17-19, 21, 30> (<7> cell-free synthesis in mRNA-dependent rabbit reticulocyte lysate system [40] <2,4,5> high activities in tissues where turnover of energy from adenine nucleotides is great, e. g. muscle [3] <1-6,11,14,15> tissue distribution [3,46] <2,5> rabbit and human carry a minimum of 2 sets of isozymes within an individual one set in muscle, erythrocytes, brain and another in liver, kidney and spleen [3]) [3, 40, 46]... 

Watanabe, K. Kubo, S. Mitochondrial adenylate kinase from chicken liver. Purification characterization and its cell-free synthesis. Eur. J. Biochem., 123, 587-592 (1982)... 

G Banko, S Wolfe, AL Demain. Cell-free synthesis of delta-(L-alpha-aminoadipyl)-L-cysteine, the first intermediate of penicillin and cephalosporin biosynthesis. Biochem Biophys Res Commun 137 528-535, 1986. 

Although cell-free synthesis of 6-dEB was achieved, kinetic studies demonstrated that the process was very inefficient [34], The low rate of biosynthesis was likely due to the fact that the association of the three DEBS proteins in vitro was suboptimal. To simplify in vitro synthesis and to facilitate mechanistic analysis, a truncated version of the erythromycin PKS was created. The protein, DEBS 1-TE, was engineered by relocating the thioesterase (TE) from the end of DEBS 3 to the terminus of DEBS 1 (Fig. 9a,b) [35], In vivo, this bimodular construct synthesizes two triketide lactone products, one derived from propionate as a starter unit, and the other from acetate. 

R Pieper, G Luo, DE Cane, C Khosla. Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases. Nature 378 263-266, 1995. 

H Peeters, R Zocher, N Madry, PB Oelrichs, H Kleinkauf, G Kraepelin. Cell-free synthesis of the depsipeptide beauvericin. J Antibiot 36 1762-1766, 1983. 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

Effect of Inhibition of Acetyl-CoA Formation on Ethyl Acetate Accumulation. Ethyl acetate has been shown to be formed from acetic acid and ethanol without any cofactors in S. cerevlslae suggesting an esterase mechanism of biosynthesis (V7). Other studies (18) found cell-free synthesis of ethyl acetate with ethanol and acetyl-CoA but not with acetic acid as a substrate. From the latter study it was proposed that ester formation in yeasts was primarily via alcoholysis of acyl-CoA compounds. Others have also suggested acetyl-CoA as an intermediate for ethyl acetate accumulation by S. cerevlslae (19). 

He M, Wang MW (2007) Arraying proteins by cell-free synthesis. Biomol Eng 24 375-380 Hook AL, Thissen H, Voelcker NH (2006) Surface manipulation of biomolecules for cell microarray applications. Trends Biotechnol 24 471 77... 

Acquaviva, A. M., Bruni, C. B., Nissley, S. P., and Rechler, M. M., Cell-free synthesis of rat insulin-like growth factor II. Diabetes 31, 656-658 (1982). 

Cell-free synthesis is often used to express troublesome proteins. For example, it is weU-suited to the expression of toxic proteins because there is no need to consider the action of a gene product on the viability of a host cell. The open nature of cell-free methods makes them useful for preparing proteins prone to mis-folding, aggregation into inclusion bodies, and proteolytic digestion. This is because the expression reaction conditions can be readily manipulated to promote proper protein folding and inhibit proteolysis. In addition. 

In the broadest terms, cell-free synthesis offers advantages over traditional in vivo methods of recombinant protein expression, as it enables the experimentalist strictly to control conditions under which expression reactions are performed. Benefits of such an open expression system are exemplified by the fact that cell-free reaction conditions can be sufficiently modified to support co-translational folding of active disulfide-bonded proteins. 

DNA template preparations are a major source of contamination in cell-free synthesis. In vitro translation reactions are sensitive to contamination by salts, RNases, detergents, and alcohol, all of which are typically used in commercial plasmid purification kits. As such, plasmids prepared by resin-based purification protocols must be further purified by phe-nol/chloroform and chloroform extraction. DNA should be precipitated with isopropanol, washed in ethanol, dried, and taken up in RNase-free water to a concentration of about 1 mg mL . The DNA should be stored frozen, in small aliquots. 

Such excellent outcomes are a result of careful and systematic optimization of DNA constructs and expression conditions. There are occasions, however, where proteins fail to express, or are expressed in an inactive form. Other proteins express well in one expression system, but not in another, and this is of course also trae of aU in vivo expression systems. A true benefit of cell-free synthesis over in vivo methods is that the open nature of the systems presents the researcher with unparalleled opportunities to affect the outcome of an expression experiment. As such, cell-free synthesis methods represent an invaluable addition to any laboratory investigating the expression of biopharmaceuticals. 

Roberts, J.L. and Herbert, E. ( 911a) Characterization of a common precursor to corticotrophin and beta-lipotrophin cell-free synthesis of the precursor and identification of corticotrophin peptides in the molecule. Proc. Natl. Acad. Sci. 74 4826 830. 

D. Mohnen Cell-free synthesis of pectin Identiflcation and partial characterization of polygalacturonate4-a-galacturonosyltransferase and its products from mem- 1061. 

Recently, cell free synthesis of bacitracin has been also studied in some detail (14). Therefore, we selected bacitracin for the production of secondary metabolite by immobilized whole cells. 

Kigawa T, Muto Y, Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable-isotope labeling of proteins for NMR analysis. J Biomol NMR 6 129-134... 

Ozawa K, Headlam MJ, Schaeffer PM, Henderson BR, Dixon NE, Otting G (2004) Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy. Eur J Biochem 271 4084-4093... 

Torizawa T, Shimizu M, Taoka M, Miyano H, Kainosho M (2004) Efficient production of isotopically labeled proteins by cell-free synthesis a practical protocol. J Biomol NMR 30 311-325... 

Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 43 12585-12591... 

Shi J, Pelton JG, Cho HS, Wemmer DE (2004) Protein signal assignments using specific labeling and cell-free synthesis. J Biomol NMR 28 235-247... 

The cell-free synthesis of strictosidine (79) and cathenamine (82) has been further explored, and the conditions under which these key compounds are formed have been optimized. Strains from C. roseus suspension cultures that were resistant to inhibition of their growth by various tryptophan analogues have been selected. The free tryptophan level in cells of these strains could be 30—40 times higher than in normal cells. Tryptophan at this level did not induce tryptophan decarboxylase, nor the production of alkaloids. It is to be noted, however, that stimulation of alkaloid production by tryptophan and tryptamine ° in cultures of normal cells has been reported. In the case of tryptamine the two most prominent metabolites were A/ -acetyltryptamine and JVN-dimethyltrypt-amine. 

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