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, fireflies living near the Black Sea in southern Russia, and obtained high level expression of the cloned sequences in Escherichia coli. The nucleotide sequences of two isolated clones were determined; five single base differences were observed, but none resulted in a change in the encoded amino acid residue. The cDNA encoded a protein of 548 amino acid residues. The overall amino acid sequence identity with the luciferase from Photinus pyralis, the North American firefly, was 67%, while comparison of the L. mingrelica luciferase with L. cruciata and L. lateralis, both indigenous to Japan, showed about 80% of the residues were strictly conserved. A novel overexpression system which employs the regulatory genes of the luminous bacterium Vibrio fischeri allowed growth of cultures to high cell density and high luciferase content, facilitating purification of the enzyme. Luciferase was purified to homogeneity in good yield from lysates of recombinant E. coli by ammonium sulfate fractionation and chromatography on columns of DEAE Sephadex and Blue Sepharose. The physicochemical properties of the luciferases from the available recombinant sources are significantly different and should allow detailed investigations into the mechanism of the bioluminescence reaction and the physical basis of the differences in the color of light emitted from the various enzymes.
Devine
J H
JH
Center for Macromolecular Design, Institute of Biosciences and Technology, Texas A & M University, College Station 77843.
Kutuzova
G D
GD
Green
V A
VA
Ugarova
N N
NN
Baldwin
T O
TO
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D008156:Luciferases