Protein Techniques

Abstract

Cell-free translation systems contain all the components necessary for protein synthesis: tRNAs; ribosomes; amino acids; initiation, elongation and termination factors; GTP; ATP; and magnesium. They require only the addition of a transcript to be translated. Single-stranded RNA for translation can be transcribed from a wide variety of plasmid vectors, such as pBlue- script®, which contain a multiple cloning site downstream from a powerful promoter for a bacteriophage DNA-dependent RNA polymerase. The two most common cell-free translation systems in use are the rabbit reticulocyte lysate system, and the wheat germ extract system. Of these the rabbit reticulocyte lysate system is more commonly used. The major drawback of the wheat germ system is its tendency to produce incomplete translation products due to premature termination (Shih and Kaesberg 1976). As a result, it is generally unsuitable for producing polypeptides greater than 60 kDa. One advantage of the wheat germ system is that it does not require the removal of endogenous mRNAs. Therefore, it may translate weakly-initiating mRNAs more efficiently than the reticulocyte lysate system. Recent modifications of the wheat germ system have made it more suitable for the translation of higher molecular weight proteins (Morch et al. 1986).