Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus.
Protein Expr Purif
; 95: 259-64, 2014 Mar.
Article
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| MEDLINE
| ID: mdl-24480186
ABSTRACT
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.
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MEDLINE
Asunto principal:
Bacillus cereus
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Proteínas Bacterianas
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Proteínas Recombinantes de Fusión
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Hidrolasas de Éster Carboxílico
Idioma:
En
Año:
2014
Tipo del documento:
Article