Abstract
We have prepared an affinity column for the purification of orotidine-5'-phosphate decarboxylase from yeast. The column effects a 3200-fold purification from yeast homogenate in one pass; simple additional steps produce enzyme that has been purified 6700-fold and is not contaminated by any other protein that can be detected by sodium dodecyl sulfate-acrylamide gel electrophoresis. Overall, 35% of the activity present in the yeast is recovered as pure enzyme. The resin for the column is synthesized by attaching the ethylenediamine amide of 5-(2-carboxyethyl)-6-azauridine 5'-phosphate to carboxymethyl-agarose.
Highlights
We have prepared an affinity column for the purification of orotidine-5’-phosphate decarboxylase from yeast
The column effects a 3200-fold purification from yeast homogenate in one pass; simple additional steps produce enzyme that has been purified 6700-fold and is not contaminated by any other protein that can be detected by sodium dodecyl sulfate-acrylamide gel electrophoresis
35% of the activity present in the yeast is recovered as pure enzyme
Summary
We have prepared an affinity column for the purification of orotidine-5’-phosphate decarboxylase from yeast. The water was removed by rotoevaporation and the trimethyl phosphate with ether; the residual solid was dissolved in 250 ml of water, the pH was adjusted to 8.5 with sodium hydroxide, and barium acetate The washed solid was dissolved in water by mixing it with cation exchange resin (35 ml; Bio-Rad AG50-X8, Na’ form); the solution was applied to a 350-ml column of the same resin and Compound IX was eluted with water.
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