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(λDE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of ˜50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as RoundupTM. 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.