Purification of isoleucyl transfer ribonucleic acid synthetase by affinity chromatography on blue dextran-sepharose.

Abstract

The aminoacyl-tRNA synthetases are a family of twenty enzymes that function in the first step of protein biosynthesis by esterifying the twenty amino acids to their cognate tRNAs [l] , Purification procedures for these enzymes are generally long and laborious. Recently several affinity chromatographic methods have been reported. Several of the aminoacyltRNA synthetases have been purified utilizing columns of purified, immobilized tRNA [2-5 ] . A method for purification of isoleucyl-tRNA synthetase (IRS) involves the use of Lisoleucinyl 5’-adenylate, an aminoacyl-AMP analog, as the insoluble ligand [6]. Unfortunately, the preparation of these column materials is generally laborious, often expensive, and may yield chromatographic media of limited stability suitable only for small scale preparations. Stellwagen [7] has reported that blue dextran linked to Sepharose may be used as an affinity chromatographic medium for many proteins which bind dinucleotides and ATP. Because the material is easily synthesized and stable, we have studied its potential as an affinity chromatographic medium for the purification of aminoacyl-tRNA synthetases. We now report the purification of IRS of Escherichia coli (EC 6.1.1.5) to near-homogeneity from crude cellular extracts by chromatography on two sequential blue dextranSepharose columns.