Translation in vitro

Wood, K. V., De Wet, J. R., Dewji, N., and DeLuca, M. (1984). Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem. Biophys. Res. Commun. 124 592-596. 

Mologni L., Lecoutre P., Nielsen P.E., Gambacorti-Passerini G. Additive anti-sense effects of different PNAs on the in vitro translation of the PML/RAR-alpha gene. Nucleic Acids Res. 1998 26 1934-1938. 

This has been estabhshed by experiments at the whole-animal level (eg, hepatectomy) and by use of the isolated perfused Hver preparation, of hver slices, of liver homogenates, and of in vitro translation systems using preparations of mRNA extracted from liver. However, the y-globulins are synthesized in plasma cells and certain plasma proteins are synthesized in other sites, such as endothelial cells. 

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.

In order to know if the presence of mature proteins correlated with the presence of mRNA, two differents experiments were carried out. In vitro translation of total mRNA isolated from mycelia growing in inducing and noninducing conditions showed similar patterns in both situations except for two bands present only in the former, between 29 and 33 kDa (Fig-4). These bands could correspond to the polypectate lyases. The patterns of both situations showed a common band, more intense in micelia grown in pectin, near 50 kDa wich could correspond to the deglicosilated PG of FORL with a Mr of 50 kDa. 

Figure 4. In vitro translation pattern of RNA. Lanes 2 and 4 from apple pectin...

Kawakami K, Salonga D, Omura K, Park JM, Danenberg KD, Watanabe Y et al. Effects of polymorphic tandem repeat sequence on the in vitro translation of messenger RNA. Proc Am Assoc Cancer Res 1999 40 436 137. 

Compounds not eliminated by the counterscreen are re-tested in a secondary assay involving in vitro translation of FF/HCV/Ren mRNA in Krebs-2 extracts using 35S-methionine followed by SDS-PAGE analysis to monitor protein synthesis... 

A general flowchart is presented in Fig. 13.5B that we followed for identifying and sorting inhibitors of translation. Shown below is an in vitro translation protocol tailored for ten 96-well assay plates (800 compounds), which can be scaled up or down as required. Negative and positive controls are present in wells A1 to D1 and El to HI, respectively. Compounds are added to wells A2 to Hll. Column 12 is left blank and could be used for additional controls, if desired. 

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. 

Scup, Stenotomus chrysops injected intraperitoneally with 1,5, or 10 mg/kg BW of PCBs 77, 105, 118, 128, or 138 and examined tor increases in ethoxyresorufin O-deethylase (EROD) activity, immunodetectable cytochrome P-450E (the EROD catalyst in scup) and in vitro translatable mRNA for P-450E... 

Flurkey WH. Polyphenoloxidase in higher plants immunological detection and analysis of in vitro translation products. Plant Physiol 1986 86 614-618. 

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

Van Gelder Was that the intensity of light used for the in vitro translated reconstitution experiment ... 

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. 

FluoroTect Green in vitro Translation Labeling System (Promega) Store at -80°C. 

The Transdirect insect cell kit is an in vitro translation system for mRNA templates. We developed and optimized a method to prepare the insect cell extract, the concentrations of the reaction components, and an expression vector pTDl (2, 5). The pTDl... 

Generally, it is difficult to detect synthesized proteins by CBB staining. To confirm the expression of the target protein, we usually perform fluorescent labeling of the in vitro translation products using the FluoroTect Labeling System (FluoroTect) (reeNote 22). 

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