Typically, translation by wheat germ extracts is more cap-dependent than translation by retic extracts. When using RNA synthesized in vitro, the presence of a 5' cap structure may enhance translational activity. Both retic and wheat germ extracts translate RNA isolated from cells and tissue or those generated by in vitro transcription (see Figure 1). The wheat germ extract is recommended for translation of RNA containing small fragments of double-stranded RNA or oxidized thiols, which are inhibitory to the rabbit reticulocyte lysate. Wheat germ lysate efficiently translates exogenous RNA from a variety of different organisms, from viruses and yeast to higher plants and mammals. This extract has low background incorporation due to its low level of endogenous mRNA. Wheat germ extract is a convenient alternative to the rabbit reticulocyte lysate cell-free system. Exogenous proteins are synthesized at a rate close to that observed in intact reticulocyte cells. This type of lysate is the most widely used RNA-dependent cell-free system because of its low background and its efficient utilization of exogenous RNAs even at low concentrations (Figure 1). Ambion offers a nuclease-treated reticulocyte lysate. The endogenous globin mRNA can be eliminated by incubation with Ca2+-dependent micrococcal nuclease, which is later inactivated by chelation of the Ca2+ by EGTA. These immature red cells have already lost their nuclei, but contain adequate mRNA, as well as complete translation machinery, for extensive globin synthesis. In vivo, reticulocytes are highly specialized cells primarily responsible for the synthesis of hemoglobin, which represents more than 90% of the protein made in the reticulocyte. Rabbit reticulocyte lysate is a highly efficient in vitro eukaryotic protein synthesis system used for translation of exogenous RNAs (either natural or generated in vitro). Each of these systems is discussed below. Standard translation systems, such as reticulocyte lysates and wheat germ extracts, use RNA as a template whereas "coupled" and "linked" systems start with DNA templates, which are transcribed into RNA then translated. There are two approaches to in vitro protein synthesis based on the starting genetic material: RNA or DNA. coli lysate), and other co-factors (Mg2+, K+, etc.). To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and Escherichia coli. This article will explain different approaches to in vitro protein synthesis (translation of purified RNA versus "linked" and "coupled" transcription:translation) and will also describe basic differences between eukaryotic and prokaryotic cell-free systems. In general, these systems are derived from cells engaged in a high rate of protein synthesis. In practice, only a few cell-free systems have been developed for in vitro protein synthesis. In principle, it should be possible to prepare a cell-free extract for in vitro translation of mRNAs from any type of cells. The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. The in vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications, including the rapid identification of gene products (e.g., proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies.
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