Role of mammalian RNase inhibitor in cell-free protein synthesis

Abstract

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.