Microgram quantities of ornithine decarboxylase (ODC, EC 184.108.40.206)-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.
Research Article|July 01 1987
Ornithine decarboxylase production in vitro by using mouse cDNA
J R Glass;
J J Duffy;
Biochem J (1987) 245 (1): 127-132.
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J R Glass, M MacKrell, J J Duffy, E W Gerner; Ornithine decarboxylase production in vitro by using mouse cDNA. Biochem J 1 July 1987; 245 (1): 127–132. doi: https://doi.org/10.1042/bj2450127
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