LOW-TEMPERATURE CULTIVATION OF ESCHERICHIA-COLI CARRYING A RICE LIPOXYGENASE L-2 CDNA PRODUCES A SOLUBLE AND ACTIVE ENZYME AT A HIGH-LEVEL

Using at T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37°C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15°C for 16 h, active lipoxygenase. amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme. © 1990.