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Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein

Authors :
Claude Sauter
Bernard Lorber
Anne Neuenfeldt
Marie Sissler
Agnès Gaudry
Catherine Florentz
Centre National de la Recherche Scientifique (CNRS)
Source :
Protein Engineering, Design and Selection, Protein Engineering, Design and Selection, Oxford University Press (OUP), 2012, 25 (9), pp.473-481. ⟨10.1093/protein/gzs046⟩
Publication Year :
2012

Abstract

International audience; Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.

Details

ISSN :
17410134 and 17410126
Volume :
25
Issue :
9
Database :
OpenAIRE
Journal :
Protein engineering, designselection : PEDS
Accession number :
edsair.doi.dedup.....7ffc24d9d1e0e46412f43e54cff300d6
Full Text :
https://doi.org/10.1093/protein/gzs046⟩