Preparation and properties of an improved cell-free protein synthesis system from mammalian liver

Abstract

A cell-free protein synthesis system derived from mouse liver has been developed which faithfully translates endogenous and exogenous mRNA. The system is based on the unfractionated post-mitochondrial supernatant. The main measures taken to improve the activity of the system were: (i) the use of high levels (30 mM) of creatine phosphate as an energy-generating system to counteract a hyperactive nucleoside triphosphatase activity in the extracts, (ii) the choice of homgenisation buffer, and (iii) the use of potassium acetate rather than KCl in the assay. The system exhibits a high initial rate of amino acid incorporation, and reinitiates translation on endogenous mRNA. Added tobacco mosaic virus (TMV) RNA is faithfully translated into full-length products at a rate of 60–80 amino acid residues per min at 30°. The rate of overall amino acid incorporation slows after about 20 min and eventually ceases due to a failure in the re-initiation of translation, and not because of degradation of mRNA. Over a limited period of time, this improved cell-free translation system is comparable in activity to other eukaryotic systems generated to date, and should be useful in studies of the control of translation rates in mammalian liver.