Ornithine decarboxylase production in vitro by using mouse cDNA

Abstract

Microgram quantities of ornithine decarboxylase (ODC, EC 4.1.1.17)-specific mRNA were synthesized by transcription techniques in vitro, by using a plasmid containing mouse cDNA coding for this enzyme. The homogeneous RNA preparation was then used for cell-free synthesis of ODC protein, in rabbit reticulocyte lysates. Analysis of products translated in vitro by polyacrylamide-gel electrophoresis revealed predominantly one protein produced, with Mr approx. 54,000, which was immunoprecipitable by anti-ODC serum. Two-dimensional gel-electrophoretic analysis showed that the protein ODC synthesized in vitro had a pI of approx. 5.4, similar to the native enzyme isolated from mouse tissues. In addition, quantification of activity and protein amount showed that the enzyme synthesized in vitro had a specific activity of approx. 63,000 units (nmol/min)/mg, consistent with the purified mouse kidney enzyme's specific activity of approx. 47,000 units/mg. An average of nearly 200 pg of ODC protein was produced in vitro from various RNA preparations. These data demonstrate that ODC-specific mRNA and active ODC protein can be produced by 'in vitro' technology, which should prove useful in studying functional and structural characteristics of these molecules.