In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System
The wheat germ extract in vitro translation system has been used widely for faithful and efficient translation of viral and eukaryotic messenger RNAs in a heterologous cell-free system (1, 2, 3, 4, 5, 6, 7, 8, 9). With respect to the yield of translation products, the wheat germ extract is less efficient than most reticulocyte lysate cell-free systems. There are advantages however of using wheat germ extracts. Firstly, the in vivo competition of mRNAs for translation is more accurately represented, making the wheat germ system preferable for studying regulation of translation (1). Secondly, particularly low levels of endogenous mRNA and the endogenous nuclease activity (14) obviate the requirement for treatment with a calcium-activated nuclease. There is therefore less disruption of the in vivo situation and contamination with calcium ions is less harmful. The identification of all sizes of exogenous mRNA-directed translation products is facilitated because of the low levels of endogenous mRNA present. Thirdly, there is no post-translational modification of translation products; primary products are therefore investigated, although processing may be achieved by the addition of microsomal membranes to the translation reaction. Fourthly, the ionic conditions of the reaction may be altered to optimize the translation of large or small RNAs (2) (see Note 1). Translational activity is optimized by the incorporation of an energy-generating system of ATP, GTP, creatine phosphate, and creatine kinase (3). Wheat germ is inexpensive and commercially available (see Note 2); preparation of the extract is rapid and simple, resulting in high yields. Wheat germ extract cell-free system kits are also commercially available.
KeywordsCreatine Kinase Wheat Germ Translation Product Translation System Microsomal Membrane
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