Purification of Aminoacyl-tRNA by Affinity Chromatography on Immobilized Thermus thermophilus EF-Tu·GTP

✍ Scribed by S. Ribeiro; S. Nock; M. Sprinzl

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 516 KB
• Volume: 228
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1359

✦ Synopsis

Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu( (\left.\mathrm{CHis}{6}\right)), was used for purification of aminoacyltRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography. Preformed aminoacyltRNA.EF-Tu (\left(\mathrm{CHis}{6}\right)).GTP ternary complexes were immobilized on (\mathrm{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 (\mathrm{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. 1995 Academic Press, Inc.