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.
Savopoulos
J W
JW
GlaxoSmithkline Pharmaceuticals, New Frontiers Science Park (North), Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom.
Hibbs
M
M
Jones
E J
EJ
Mensah
L
L
Richardson
C
C
Fosberry
A
A
Downes
R
R
Fox
S G
SG
Brown
J R
JR
Jenkins
O
O
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