The simple format of the PURE system allows it to be easily integrated into high throughput platforms for functional genomics and proteomics studies. The absence of any nuclease activities ensures the stability of linear DNA templates during protein synthesis. Individual DNA templates for in vitro expression can be generated by PCR, eliminating the time-consuming cloning process. This feature is particularly useful for high throughput screening at the whole genome scale, either for novel activities or for protein-protein interactions. For structural genomics projects, the PURE system can be an alternative route to acquire difficult protein targets which resist traditional cellular expression (4).
Since the early pioneering work of Nirenberg and Matthaei in 1961 (1), which demonstrated in vitro protein translation using cell extracts, cell-free protein synthesis has become an important tool for molecular biologists by playing a centn Dr. Takuya Ueda’s lab at the University of Tokyo. This became known as the “PURE” system, which stands for “Protein synthesisral role in a wide variety of applications (2). In the post-genomic era, cell-free protein synthesis has the potential to become one of the most important high throughput technologies for functional genomics and proteomics.
The synthesis of proteins from RNA is known as translation
The information required to build resides on the in the form of sequences of , and has the form of triplets of bases called "codons" following the . Once the genetic information describing how to build a is to , it has to be "translated" into the "language" of the protein. The sequence of on the RNA forms a code for the building blocks of the protein. The word "translation" seems particularly appropriate for this process since the information contained in the DNA is translated from the four-character alphabet of the bases to the twenty-character alphabet of the amino acids.
Protein synthesis by pure translation systems - …
Commercially available cell-free protein synthesis systems are typically derived from cell extracts of Escherichia coli S30, rabbit reticulocytes or wheat germ. The drawback of extract-based systems is that they often contain nonspecific nucleases and proteases that adversely affect protein synthesis. In addition, the cell extract is like a “black box” in which numerous uncharacterized activities may modify or interfere with subsequent downstream assays.
Protein Synthesis: Transcription and Translation
PURExpress™ from NEB is based on the PURESYSTEM® from PGI, and improves on the original “Classic II” Kit by optimizing components to increase yield of protein synthesis. For more information please .
Translation and Protein Synthesis Quiz or Review by …
The biggest advantage, compared to protein production in living cells, is that cell-free protein synthesis is the quickest way to obtain an expressed phenotype (protein) from a genotype (gene). Starting with a PCR or plasmid template, in vitro protein synthesis and functional assays can be carried out in a few hours. Moreover, it is independent of host cells. Proteins which are toxic or prone to proteolytic degradation can be readily prepared in vitro.
Control of ribosomal protein synthesis
The PURE system contains the minimal set of factors necessary for in vitro protein translation. It is largely free of chaperones and other cellular factors for post-translational modifications, thus providing a starting point to study the involvement of these factors in transcription/translation regulation and nascent chain folding (10). It can also be used to produce “clean” proteins which, if purified from traditional cellular hosts, may come with undesired modifications or bound co-factors. A number of research labs studying translation routinely use home-made reconstituted systems to study different aspects of translation.