Coupled transcription/translation

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

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. 

Coupled transcription-translation in prokaryotes refers to the commencement of translation of an RNA molecule before its transcription from the DNA template is complete. Could such a situation arise in eukaryotes ... 

The mRNA required for in vitro translation can itself be produced by in vitro synthesis. Commercially available kits allow DNA cloned downstream of T7-, T3 or SP6-promoters to be transcribed effectively in vitro by the relevant RNA polymerases. In coupled transcription-translation, it should be remembered that translation of eukaryotic mRNA requires a 5 cap upstream of the initiation codon, and similarly, for prokaryotic translation there should be an appropriately positioned ribosome binding site. Commercial kits are also available for combined in vitro transcription and translation. 

Two genes (psbE and psbF) for polypeptides of Cyt b-559 have been,located and characterized from spinach [56], wheat [57], tobacco [58], Oenothera hookeri [58] and pea (D.L. Willey, unpublished). The gene for the 9 kDa polypeptide known to be associated with Cyt b-559 preparations was first of all located in spinach [59] and wheat [57] by hybrid-release translation or by coupled transcription-translation of fragments of chloroplast DNA. Nucleotide sequencing [56,57] revealed an open reading frame of 83 codons, encoding a protein with the known N-... 

Before embarking on a particular expression strategy it is useful to have some information regarding the hands-on time required before the first results can be obtained. The fastest turnaround times are achieved with cell lysates, such as the reticulocyte and wheat germ lysates, which only need to be primed with in vitro synthesized RNA (or DNA in the case of a coupled transcription/translation system). These systems potentially provide answers within a few hours but are rarely used because of the small quantities that they yield. Plasmid-based systems used for expression in E. coli, yeast, and in combination with the vaccinia-T7 vector system are relatively fast as well. These strategies require subcloning of the gene(s) of interest into a plasmid vector, which is then transfected into the... 

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. 

Table 15.3 Conditions for coupled transcription/translation systems from . coli under reducing and oxidizing conditions...

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-... 

Kim DM, Choi CY (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol Prog 12 645-649... 

As the E. coli CF setup is a coupled transcription/translation system, DNA is required as template. Insufficient (or no) production of a target protein is caused in most cases by problems with the central transcription/translation process. Appropriate template design and purity are thus crucial for successful CF expression. For screening and high-throughput purposes, linear templates generated by PCR may be used the more stable circular plasmid DNA templates are recommended for preparative scale expression. 

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

Generation of ARM complexes by coupled transcription/translation in vitro... 

A number of in vitro transcription/translation systems are commercially available, including rabbit reticulocyte, wheat germ, and E. coli S30 extracts (18). There is also a variety of rabbit reticulocyte sj tems, including nuclease-treated, non-nuclease-treated, DTT-deficient, and coupled transcription/translation. We... 

TNT T7 Quick coupled transcription/ translation system (Promega, Cat. No,... 

Set up in vitro coupled transcription/translation mixture in a siliconized tube. ... 

Construction of fiagments from hybridomas and lymphocyte mRNA 95 Generation of ARM complexes by coupled transcription/translation in vitro 97 Preparation of antigen- or streptavidin-conpled magnetic beads 98 Antigen selection of ARM complexes 99... 

Ohuchi S, Nakano H and Yamane T (1998) In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/ translation. Nucleic Adds Res 26 4339—4346. 

P450cam expressed in Escherichia coli. c)P450cam synthesized in vitro using Escherichia coli S30 coupled transcription /translation system. ND not detected. 

NS proteins. Complete replication of full-length plus- and minus-strand nucleocapsids has been achieved in a cell-free coupled transcription-translation reaction in the presence of a required cellular factor(s) (Hill et al., 1981 Patton et ai, 1983). In fact, Wertz (1983) has been able to replicate the RNA of defective-interfering VSV nucleocapsids in a cell-free system coupled with VSV mRNA translation but free of infectious full-length nucleocapsids. 

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