Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ

Abstract

A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of (14)C leucine incorporated into protein in a 50 mul assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg(+2), K(+) and spermine concentration as well as the method of extract preparation. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.