Cell-free transcription/translation system

The ability of both suppressor tRNAs to incorporate the nonpolar amino acid valine as well as the polar noncoded homoglutamate into two proteins was tested in E. coli cell-free transcription-translation systems [35]. The proteins T4... 

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... 

Transcription of RNA from DNA is far simpler than translation of RNA into protein, and large RNA molecules can be synthesised using a simple in vitro cell free transcription system (see Chapter 2). Typically, the DNA coding for the RNA of interest is inserted into pDNA downstream of a promoter such as the T3, T7 or Sp6 promoter. RNA is then synthesised from the pDNA in vitro using the most appropriate DNA-dependent RNA polymerase for the promoter (i.e., T3 RNA polymerase, T7 RNA polymerase or Sp6 RNA polymerase). 

In the case of post-transcriptional control the information gap is marginally better. Recent technical advances (for example, in the isolation of specific mRNA s and their translation in cell-free protein synthesizing systems and in the field of RNA-DNA hybridization) have begun to improve our understanding of exactly what happens to a particular mRNA after it has been transcribed and how its utilization may be controlled. It is the aim of this review to outline some of this new information and the light it sheds on the problem of the regulation of gene expression in animal cells. 

Capped messengers are more readily translated than uncapped ones in eukaryotic cell-free protein synthesis systems removal of the caps interferes with tire binding of the mRNA to the ribosome. It is not known when the cap is applied in vivo, but it may be part of the initiation of transcription, as capped mRNA fragments can be isolated from cells in which elongation of the messenger has been inhibited. [A.J.Shatkin et al. in Messenger RNA and Ribosomes in Protein Synthesis, C.F.Phelps H.R.V.Ainstein, eds. (The Biochemical Society, London, Symposium 47) 19. ]... 

Kigawa T, Yokoyama S (1991) A continuous cell-free protein synthesis system for coupled transcription-translation. J Biochem 110 166-168... 

A protein microarray relies on high-throughput amplification of each predicted ORF by using gene-specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system. The protein products from the unpurified reactions are printed directly onto nitrocellulose microarrays without further purification [113]. 

On the basis of our previous results discussed above, early viral transcripts were primary candidates in the inhibition of host protein synthesis. Since in vitro transcription produces only the early species of viral transcripts (Kates and Beeson, 1970), we acquired these early species of viral RNA by means of in vitro transcription by viral cores and tested their effect on the translation of various exogenous mRNAs in in vitro cell-free systems rendered messenger dependent. The results from such experiments (Coppola and Bablanian, 1983) revealed that transcripts prepared in vitro of either 8-10 S or 4-7 S size classes inhibit globin, HeLa, and hamster cell mRNA translation in a reticulocyte cell-free protein-synthesizing system. Inhibition was observed not only under conditions where in vitro viral transcripts by themselves were capable of producing viral polypeptides, but also when low concentrations of in v/7ro-synthesized transcripts were used which were incapable of synthesizing polypeptides as determined by polyacrylamide-gel electrophoresis. In contrast, the transcripts synthesized in vitro by vaccinia virus cores had no inhibitory effect on the translation of cytoplasmic RNA obtained from vaccinia virus-infected cells at early times after infection. Therefore, this inhibitory effect, like that seen in vaccinia virus-infected cells, was selective. The vaccinia virus transcripts, synthesized in vitro, also inhibited encephalomyocarditis virus mRNA translation in the cell-free reticulocyte lysate system indicating that this inhibition does not require... 

Pelham, H. R. B., Sykes, J. M. M., and Hunt, T., 1978, Characteristics of a coupled cell-free transcription and translation system directed by vaccinia cores, Eur. J. Biochem. 82 199. 

A cell-free extract (S30) from E. coli K12 has been developed as an efficient coupled transcription/translation system which performs protein synthesis in vitro from the genes cloned in plasmids under the T7, T3, or SP6 promoter. Capping of the mRNA for eukaryotic proteins is apparently unnecessary with the S30 system. The coupled transcription/translation system, which was originally developed as the S3 0 translation system (122), can be used in a batchwise or continuous-flow mode (123,124). Use of the ribosome fraction collected from the S30 extracts is reported to improve the yield and efficacy further and more advantageously with nonlinearized plasmids than with linearized plasmids (125). 

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

Fig. 4. Massive production of green fluorescent protein (GFP) by the continuous-flow cell-free method. Sodium dodecyl sulfide-polyacrylamide gel electrophoresis analysis of GFP produced during 14 d of reaction. mRNA produced by transcription of circular plasmid of Ehime University (pEU) was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1 -pL aliquot of the mixture was run on the gel and protein bands were stained with Coomassie Brilliant Blue. The arrow shows GFP and st designates an authentic GFP band (0.5 pg).

Automating the translation steps, starting with transcription in the wheat germ cell-free translation system (improved as above) using robotics (26)... 

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

Transcription and translation are coupled in the cytoplasm of prokaryotes. In eukaryotes, transcription of DNA and subsequent processsing of the messenger RNA transcript both occur in the nucleus, while translation of the mature mRNA template occurs in the cytoplasm. Cell-free systems lack mechanisms for mRNA processing, and therefore require fully matured... 

The conditions for performing a wheat embryo cell-free translation reaction from an mRNA template are listed in Table 15.4. Points discussed for E. coli optimization in Section 15.6.1 are relevant to expression in wheat embryo extracts. However, it is worth noting that the wheat embryo system is better suited to the translation of added mRNA template, whereas coupled transcription/translation is better in the E. coli system. The principal reason for this difference is that transcription with bacteriophage RNA polymerases requires a relatively high Mg concentration (ca. 16 mM) E. coli translation-only reac-... 

Ribosome Display. This cell-free display system uses the same principle as the phage-display systems in bacteria. The phenotype and genotype of a peptide are linked together and, therefore, can be simultaneously selected based on the function of the peptide. However, the linkage is a physical one in that the genetic material is covalently linked to its encoded product in the formation of an antibody-ribosome-mRNA (ARM) complex through in vitro transcription/translation [59]. 

Although interferons are mediators of immune response, different mechanisms for the antiviral action of interferon have been proposed. Interferon-a possesses broad-spectrum antiviral activity and acts on virus-infected cells by binding to specific cell surface receptors. It inhibits the transcription and translation of mRNA into viral nuoleic acid and protein. Studies in cell-free systems have shown that the addition of adenosine triphosphate and double-stranded RNA to extracts of interferon-treated cells activates cellular RNA proteins and a oellular endonuclease. This activation causes the formation of translation inhibitory protein, which terminates production of viral enzyme, nucleic acid, and structural proteins (28). Interferon also may act by blocking synthesis of a cleaving enzyme required for viral release. 

---