Luciferases

Details Luciferases Luciferases

1996-11-20

2000-10-17

Prouty, Rebecca E.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving luciferase

435 6, 435189, 435325, 435348, 4352523, 43525233, 43525421, 4353201, 435440, 536 232, C12N 902, C12N 1553, C12Q 166

Patent

active

061329835

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to novel proteins having luciferase activity and to DNA and vectors encoding for their expression. Particularly the present invention provides luciferases having heat stability at temperatures above 30.degree. C.
Firefly luciferase catalyses the oxidation of luciferin in the presence of ATP, Mg.sup.2+ and molecular oxygen with the resultant production of light. This reaction has a quantum yield of about 0.88 (see DeLuca & McElroy (1978) and Seliger & McElroy (1960)) and this light emitting property has led to its use in luminometric assays where ATP levels are being measured.
Luciferase is obtainable directly from the bodies of insects such as fireflies or glow-worms or by expression from microorganisms including recombinant DNA constructs encoding for the enzyme. Four significant species of firefly from which the enzyme may be obtained, or DNA encoding for it may be derived, are the Japanese GENJI and HEIKE fireflies Luciola cruciata and Luciola lateralis, the East European Firefly Luciola mingelica and the North American firefly (Photinus pyralis). The glow-worm Lampyris noctiluca is a further source with the amino acid sequence of its luciferase having 84% homology to that of Photinus pyralis.
The heat stability of wild and recombinant type luciferases is such that they lose activity quite rapidly when exposed to temperatures in excess of about 30.degree. C., particularly over 35.degree. C. Such instability renders the enzyme deficient when used or stored at high ambient temperatures or if heat induced increase in reaction rate is required. It is known that Japanese firefly luciferase can be stabilised against heat inactivation by mutating it at its position 217 to replace a threonine residue by an isoleucine residue (Kajiyama & Nakano (1993) Biochemistry 32 page 13795 to 13799). In this manner the thermal and pH stability and the specific activity of the enzyme were increased. The heat stabilisation of Photinus Dyralis and Luciola mingrelica luciferases has not yet been reported.
The present inventors have now provided novel luciferases having increased heat stability over wild type luciferases by replacing a glutamate residue present in a sequence conserved in each of Photinus pyralis, Luciola mingrelica, Luciola lateralis and Luciola cruciata with alternative amino acids, particularly lysine or arginine. This glutamate is found at position 354 in Photinus pyralis luciferase, at the third amino acid of the conserved amino acid sequence TPEGDDKPGA found in the luciferases of this and the other species.
Thus in the first aspect of the invention there is provided a protein having luciferase activity and having over 60% homology of amino acid sequence with that of Photinus pyralis, Luciola minzrelica, Luciola cruciata or Luciola lateralis characterised in that the amino acid residue corresponding to residue 354 of Photinus pyralis luciferase and residue 356 of Luciola mingrelica, Luciola cruciata and Luciola lateralis luciferase is an amino acid other than glutamate.
The amino acid may be a naturally occurring amino acid or may be a so called unusual amino acid such as an modified naturally occurring amino acid or an analogue of such. Analogues of amino acids other than glutamate will be understood to be those compounds that have equivalent effect on the protein to the amino acid of which they are analogues. Typical unusual amino acids are those as set out in the US and European Patentin Manuals and the Rules of Practice in Patent Cases: application disclosures containing nucleotide and/or amino acid sequences: modified and unusual amino acids.
Preferably the protein is characterised in that it comprises an amino acid sequence XGDDKPGA wherein X is the amino acid other than glutamate. More preferably the protein comprises the amino acid sequence TPXGDDKPGA and preferably, for thermostability, X is any amino acid other than aspartic acid, proline or glycine; still more preferably it is tryptophan, valine, leucine, isoleucine or asparagine but most preferably is lysine or argin

REFERENCES:
L.G. Strause et al., "Characteristices of Luciferases From a Variety of Firefly Species: Evidence For the Presence of Luciferase Isozymes" Insect Biochem. 11(4): 417-422, 1981.
F.R. Leach et al., "Cloning and Sequencing of a Firefly Luciferase From Photuris pennsylvanica", Proceedings of the 9th Internation Symposium on Bioluminescence and Chemiluminescence, J.W. Hastings et al. (eds) John Wiley & Sons, Chichester England, 240-, Oct. 1996.

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