Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase

Abstract

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.