Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase |
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Authors: | Arnaud Ducruix Nestor Hounwanou Joseph Reinbolt Yves Boulanger Sylvain Blanquet |
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Institution: | 1. Laboratoire de Biochimie, Ecole Polytechnique, 91128 Palaiseau Cedex, France;2. Institut de Chimie des Substances Naturelles du Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette France;3. Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, 15, Rue René Descartes, 67084 Strasbourg Cedex France |
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Abstract: | Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated from an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the α- and β-subunits composing the native α2β2 enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon. |
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Keywords: | Phenylalanyl-tRNA synthetase Subunit dissociation Gene cloning Plasmid Aminoacyl-tRNA synthetase (E coli) |
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