Identification, cloning, and expression of a functional phenylalanyl-tRNA synthetase (pheRS) from Staphylococcus aureus.
Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.
GlaxoSmithkline Pharmaceuticals, New Frontiers Science Park (North), Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom.
J W
JW
Savopoulos
M
M
Hibbs
E J
EJ
Jones
L
L
Mensah
C
C
Richardson
A
A
Fosberry
R
R
Downes
S G
SG
Fox
J R
JR
Brown
O
O
Jenkins
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009876:Operon / Q000235:genetics; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000201:enzymology; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology
D011994:Recombinant Proteins; D010652:Phenylalanine-tRNA Ligase