Overproduction of phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 in Escherichia coli.
The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus was overproduced in Escherichia coli. Three different promoter systems were used for the overexpression of the pheST genes: the tac, araB, and T7 promoters. Despite several attempts for improvement, the overproduction of the FRS was lower than that found with most of the other T. thermophilus genes. Nevertheless, enzyme amounts sufficient for biochemical and biophysical studies could be obtained more easily from the overproducing E. coli than from T. thermophilus, since at least fivefold higher specific FRS activity was present in the overproducing cells than in T. thermophilus. Also, a simple purification procedure was established. After heat treatment at 70 degrees C to remove thermolabile E. coli proteins, only three chromatographic steps, i.e., Q-Sepharose FF, hydroxyl apatite, and heparin-Sepharose chromatography, were necessary to obtain apparently homogeneous FRS. With a different plasmid construction we introduced six histidine residues at the N terminus of the alpha subunit. Thus, affinity chromatography on a nickel-chelate matrix can be used for the purification of FRS as well as for its mutant variants, which may be less stable than the native FRS and cannot be purified with heat treatment. We also cloned the pheST genes in a phagemid, which will enable mutagenesis studies and overexpression in a one-vector system without any subcloning steps.
Lechler
A
A
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Keller
B
B
Hennecke
H
H
Kreutzer
R
R
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D008969:Molecular Sequence Data; D009154:Mutation; D010652:Phenylalanine-tRNA Ligase / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D010652:Phenylalanine-tRNA Ligase