In vitro coupled transcription/translation

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

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. 

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... 

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... 

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. 

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). 

Enzyme synthesis in vitro supplements genetic analyses by providing information on the map positions of promotors and genes. And above all, the coupled transcription-translation systems have opened new approaches into the regulation of protein synthesis such as the understanding of the control of the lactose operon and of catabolite repression. Enzyme synthesis in vitro... 

Technically, in vitro transcription is achieved from standard expression plasmids typically carrying SP6 or T7 promoters using marketed kits. Translation into the polypeptide may be either coupled directly to the transcription (in vitro TnT) or require isolation of the RNA. Again, a large number of suitable prokaryotic and eukaryotic cell extracts as well as complementation factors are freely available. 

Mattheakis et al., 1994). A library of 1012 DNA molecules was used with E. coli ribosome display utilizing a coupled in vitro transcription-translation system. This library was selected for binding to the monoclonal antibody D32.39, which originally bound dynorphin B, a 13-residue opioid peptide, with 0.29 nM affinity. Five cycles of ribosome display resulted in several different peptides with affinities to the antibody ranging from 7.2 to 140 nM affinity. Yet, a peptide with a sequence similar to dynorphin B was not isolated. 

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