Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus.
Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [14C]tryptophan per A260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtrp were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAtrp has been crystallized under several different conditions.
D002250:Carbon Radioisotopes; D002852:Chromatography, Ion Exchange / Q000379:methods; D003460:Crystallization; D001411:Geobacillus stearothermophilus / Q000032:analysis*; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification*
D002250:Carbon Radioisotopes; D012346:RNA, Transfer, Amino Acyl