Glass J R, MacKrell M, Duffy J J, Gerner E W
Department of Radiation Oncology, University of Arizona Health Sciences Center, Tucson 85724.
Biochem J. 1987 Jul 1;245(1):127-32. doi: 10.1042/bj2450127.
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.
通过体外转录技术，利用含有编码该酶的小鼠cDNA的质粒，合成了微克量的鸟氨酸脱羧酶（ODC，EC 4.1.1.17）特异性mRNA。然后将这种均一的RNA制剂用于在兔网织红细胞裂解物中无细胞合成ODC蛋白。通过聚丙烯酰胺凝胶电泳对体外翻译产物的分析显示，主要产生一种蛋白质，其Mr约为54,000，可被抗ODC血清免疫沉淀。二维凝胶电泳分析表明，体外合成的ODC蛋白的pI约为5.4，与从小鼠组织中分离的天然酶相似。此外，活性和蛋白量的定量显示，体外合成的酶的比活性约为63,000单位（nmol/分钟）/mg，与纯化的小鼠肾酶的比活性约47,000单位/mg一致。从各种RNA制剂中体外平均产生近200 pg的ODC蛋白。这些数据表明，ODC特异性mRNA和活性ODC蛋白可以通过“体外”技术产生，这在研究这些分子的功能和结构特征方面应该是有用的。
