Expression, purification, and functional analysis of mammalian aminoacyl‐tRNA synthetases

• Published in: The FASEB journal 2007, Vol.21 (6), p.A1026-A1026
• Main Authors: Guzzo, Catherine M, Yang, David C. H.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.21.6.A1026

The aminoacyl‐tRNA synthetases are key enzymes in translation of the genetic code. In comparison to their prokaryotic homologues, aminoacyl‐tRNA synthetases from higher eukaryotes associate as multi‐synthetase complexes and contain extensions appended to the N or C terminus of the protein. The functions of the extensions have been of intense interest given that the extensions are dispensable for aminoacylation. The role of the N‐terminal extension in tRNA synthetase on the binding of tRNA, enzyme stability, substrate channeling, and multi‐synthetase complex formation was examined. Mammalian enzymes such as aminoacyl‐tRNA synthetases are frequently produced as inactive proteins in E. coli. A variety of fusion partners improve the expression and solubility of recombinant proteins. Only ubiquitin and SUMO fusion systems allow specific cleavage of the fusion partner. Selection of fusion and affinity tags is typically empirical. A systematic analysis and comparison of the expression and purification of human tRNA synthetase using ubiquitin and SUMO fusion proteins was carried out. The expression level, solubility, yield, and activity of tRNA synthetase with different fusion partners were systematically examined. The analysis should provide better understanding of the evolutionary divergence of tRNA synthetases. Improvement of the expression systems in E. coli should facilitate the production of a large number of recombinant synthetases for further studies.