The most frequently used cell-free expression systems originate from rabbit reticulocytes, wheat germ and E. coli. There are two types of rabbit reticulocyte-based cell-free expression systems: Translation Systems and Coupled Translation and Transcription (TNT®) Systems. Both provide the macromolecular components required for translation, such as ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors. Protein synthesis can be performed in a few hours using mRNA templates in translational systems or DNA template (plasmid DNA or PCR fragments) in coupled transcription and translation systems.
The T7 RNA Polymerase System is the most popular approach for producing proteins in E. coli. In this system, an expression vector containing a gene of interest, cloned downstream of the T7 promoter, is introduced into a T7 expression host. T7 expression hosts such as DE3 strains have a chromosomal copy of the phage T7 RNA polymerase gene. When an inducer such as IPTG or rhamnose is added to the culture, T7 RNA polymerase is expressed and transcribes the gene of interest, followed by translation of the desired protein by endogenous protein translation machinery. Promega offers ready–to–use competent cells for expression of recombinant proteins in E. coli.