In Vitro Translation Systems

Researchers synthesize recombinant proteins in cell-free extracts to verify the identity of cloned genes, to study protein-protein, protein-nucleic acid, and protein-drug interactions, and to carry out mutagenesis studies. In vitro protein translation studies rely on the efficient and selective transcription of cloned genes in vitro, which is now not only possible, but also routine, thanks to the identification of bacteriophage (phage) DNA-dependent RNA polymerases and their promoters.1 Phage RNA polymerases are single subunit enzymes that polymerize RNA from DNA templates at rates up to five times faster than bacterial polymerases.

Three phage RNA polymerases are generally used for in vitro transcription: Salmonella typhimurium phage SP6 RNA polymerase, Escherichia coli phage T3 RNA polymerase, and E. coli phage T7 RNA polymerase.1 These three polymerases are highly specific for their individual promoters, recognize promoter sequences not likely to occur in mammalian or bacterial DNA sequences, and...

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