[3] Cell-free translation of messenger RNA in a wheat germ system

Cell-free extracts of wheat germ embryos, containing ribosomes and soluble factors, support the in vitro translation of a wide variety of mRNAs into protein. This chapter describes methods that are used to study the in vitro synthesis of proteins, each comprise several percent of total cellular protein, such as globin, prolactin, the caseins, and to the synthesis of the lysosomal proteins cathepsin D, β-glucuronidase, and glucocerebrosidase. The wheat germ system is used widely because, it is relatively easy to prepare and contains a relatively low amount of endogenous mRNA. The low concentration of endogenous mRNA present in the wheat germ extract competes with the added RNA for ribosomes and factors required for translation; therefore, it is advantageous to reduce the concentration of endogenous RNA by treating with nuclease. The scale of the translation reaction required for detection of the protein under study depends upon the amount of its mRNA in the total RNA preparation.