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Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein
- 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.
- Subjects :
- MESH: Gene Expression
[SDV]Life Sciences [q-bio]
Aspartate-tRNA Ligase
Molecular Sequence Data
Gene Expression
Bioengineering
MESH: Amino Acid Sequence
Biology
Cleavage (embryo)
Crystallography, X-Ray
Protein Engineering
Biochemistry
MESH: Recombinant Proteins
Mitochondrial Proteins
MESH: Protein Structure, Tertiary
03 medical and health sciences
Protein structure
Escherichia coli
Humans
MESH: Aspartate-tRNA Ligase
Amino Acid Sequence
Solubility
Molecular Biology
Peptide sequence
MESH: Crystallization
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
MESH: Humans
MESH: Molecular Sequence Data
MESH: Escherichia coli
030302 biochemistry & molecular biology
MESH: Mitochondrial Proteins
MESH: Crystallography, X-Ray
Recombinant Proteins
Amino acid
Protein Structure, Tertiary
MESH: Solubility
MESH: Protein Engineering
N-terminus
Cytosol
Enzyme
chemistry
Crystallization
Biotechnology
Subjects
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⟩