Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter.
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E. coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from E. coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.
Shuttleworth
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WA
Department of Biochemistry/Biophysics, Washington State University, Pullman 99164-4660.
Hough
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CD
Bertrand
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Evans
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D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013604:T-Phages / Q000235:genetics*; D014166:Transferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D012321:DNA-Directed RNA Polymerases