Purification of transfer RNA species by single-step ion-exchange high-performance liquid chromatography

Anion-exchange high-performance liquid chromatography (HPLC) methods have been developed for the purification and concentration of milligram quantities of tRNA. A Waters Protein Pak DEAE 5PW 150 x 21.5 mm I.D. column was utilized for the separation of tRNA species. The chromatographic conditions chosen created non-denaturing conditions for separating the different species: 0.1 M Tris buffer (pH 7.6) at 25 degrees C, with a 0.25 M to 0.4 M sodium chloride gradient, using a 170-min gradient. The gradient form could be adjusted for optimizing purification (to over 85%) of the tRNA species of interest. The same DEAE packing in a smaller column was found to be effective for concentrating solutions of the purified tRNA. Fifty-fold concentration and recoveries above 90% have been obtained by this method. These methods were successfully applied to the purification of individual tRNA species from both Escherichia coli and yeast.