Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP

Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography. Preformed aminoacyl-tRNA.EF-Tu(CHis6).GTP ternary complexes were immobilized on Ni(2+)-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP. Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu's affinity for aminoacyl-tRNA. The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts.