Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.
Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.
Bonin
Paul D
PD
Cell and Molecular Biology, Rockville, Maryland, USA.
Choi
Gil H
GH
Trepod
Catherine M
CM
Mott
John E
JE
Lyle
Stephen B
SB
Cialdella
Joyce I
JI
Sarver
Ronald W
RW
Marshall
Vincent P
VP
Erickson
Laurence A
LA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D006639:Histidine; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase