LUCIFERASE FROM THE EAST EUROPEAN FIREFLY LUCIOLA-MINGRELICA - CLONING AND NUCLEOTIDE-SEQUENCE OF THE CDNA, OVEREXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION OF THE ENZYME

We have cloned cDNA encoding luciferase in Luciola mingrelica, firefli es living near the Black Sea in southern Russia, and obtained high lev el expression of the cloned sequences in Escherichia coli. The nucleot ide sequences of two isolated clones were determined; five single base differences were observed, but none resulted in a change in the encod ed amino acid residue. The cDNA encoded a protein of 548 amino acid re sidues. The overall amino acid sequence identity with the luciferase f rom Photinus pyralis, the North American firefly, was 67%, while compa rison of the L. mingrelica luciferase with L., cruciata and L. lateral is, both indigenous to Japan, showed about 80% of the residues were st rictly conserved. A novel overexpression system which employs the regu latory genes of the luminous bacterium Vibrio fischeri allowed growth of cultures to high cell density and high luciferase content, facilita ting purification of the enzyme. Luciferase was purified to homogeneit y in good yield from lysates of recombinant E. coli by ammonium sulfat e fractionation and chromatography on columns of DEAE Sephadex and Blu e Sepharose. The physicochemical properties of the luciferases from th e available recombinant sources are significantly different and should allow detailed investigations into the mechanism of the bioluminescen ce reaction and the physical basis of the differences in the color of light emitted from the various enzymes.