Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus.
Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in Escherichia coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis-Menten parameters reported.
Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. Electronic address: kdtcreel@gmail.com.
Kasey
K
Taylor-Creel
Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. Electronic address: marychames@gmail.com.
Mary C
MC
Hames
Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. Electronic address: wbholloway2@gmail.com.
W Blake
WB
Holloway
Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. Electronic address: hk0003@uah.edu.
Hana
H
McFeeters
Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. Electronic address: robert.mcfeeters@uah.edu.
Robert L
RL
McFeeters
D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D012995:Solubility
D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase
Kinetic parameters; Peptidyl-tRNA hydrolase; Purification; Recombinant expression; Solubility optimization