Preparation and properties of an improved cell-free protein synthesis system from mammalian liver.
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: 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, the choice of homgenisation buffer, and 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 degrees. 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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051379:Mice; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000235:genetics
D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D012367:RNA, Viral; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate