Mutant luciferases

Details Mutant luciferases Mutant luciferases

1997-10-01

2001-01-09

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving luciferase

C435S189000, C435S320100, C435S252300, C435S252330, C435S254210, C536S023200

Reexamination Certificate

active

06171808

ABSTRACT:

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 lower K
m
for the substrate ATP than existing native and recombinant luciferases of wild and altered wild type.
Firefly luciferase catalyses the oxidation of luciferin in the presence of ATP, Mg
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 bodies of fireflies or by expression from microorganisms including recombinant DNA constructs encoding for the enzyme. Significant species from which the enzyme may be obtained, or DNA encoding for it derived, are the Japanese GENJI and HEIKE fireflies
Luciola cruciata
and
Luciola lateralis,
the East European Firefly
Luciola mingrelica,
the North American firefly (
Photinus pyralis
) and the glow-worm and the European glow-worm
Lampyris noctiluca.
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° C., particularly over 35° C., and this renders the enzyme deficient when used at high ambient temperatures. It is known that Japanese firefly luciferase can be heat stabilised by mutating it at its position 217 to replace a threonine residue by an isoleucine residue (Kajiyama and Nakano (1993) Biochemistry 32 page 13795 to 13799); pH stability and specific activity also being increased.
Copending patent application GB 9405750.2 discloses an amino acid substitution that is capable of increasing the thermostability of inter alia,
Photinus pyralis
which can be used with the change at 217 to provide luciferase that is relatively heat stable at 50° C. or more.
The present invention relates to a further enhancement of the properties of luciferase enzymes, making them suitable for use in assays based upon the detection of adenosine triphosphate at relatively low levels. This enhancement is provided by changing the amino acid at the position corresponding to position 270 in the
Photinus pyralis
luciferase amino acid sequence whereby the Michaelis-Menten constant (K
m
) of the enzyme is decreased as compared to a corresponding luciferase having wild-type sequence. This corresponds to amino acid 272 in
Luciola mingrelica, Luciola cruciata
and
Luciola lateralis.
It also corresponds to amino acid 270 in
Lampris Noctiluca.
The present enhancement further provides luciferases that are characterised by the ability to oxidise D-luciferin with light emission of a different wavelength to that of wild-type luciferase, thus allowing them to be used as specific labels in binding assays wherein the wavelength of light emitted is characteristic of a particular labelled material being present, or allows DNA encoding for the luciferases to be used as a reporter DNA for genetically engineered cells and cells derived therefrom.
Thus in the first aspect of the present invention there is provided a protein having luciferase activity and having over 60% homology of amino acid sequence with that of
Photinus pyralis, Luciola mingrelica, Luciola cruciata
or
Luciola lateralis
characterised in that the amino acid residue corresponding to residue 270 of
Photinus pyralis
luciferase and residue 272 of
Luciola mingrelica, Luciola cruciata
and
Luciola lateralis
luciferase is an amino acid other than glutamate. Preferably is characterised in that it comprises a conserved amino acid sequence F(1)XE(2)FL wherein (1) is D or E, (2) is T or L and X is the amino acid other than glutamate; F, E, L, D and T each relating to the corresponding amino acid as provided for by the single letter amino acid code.
The preferred amino acid X so far determined is lysine, or an analogue or modification thereof. Other preferred amino acids include arginine, glutamine and alanine.
In still more preferred forms of the present invention the protein of the invention also has the amino acid at the position corresponding to amino acid 217 of the Luciola firefly luciferases or 215 of
Photinus pyralis
changed to a hydrophobic amino acid, preferably to isoleucine, leucine or valine or analogue or these and/or has the amino acid at the position corresponding to amino acid 356 of the Luciola firefly luciferase or 354 of
Photinus pyralis
changed to an amino acid other than glutamate, particularly to lysine, arginine, leucine, isoleucine or histidine or analogues or modifications of these.
In a second aspect of the invention there is provided DNA encoding for the protein of the invention and in a third aspect there is provided a vector, particularly a plasmid, comprising a luc gene (the gene encoding for luciferase) in such a form as to be capable of expressing the protein of the invention. Such forms are those where the vector includes DNA sequences capable of controlling the expression of the protein of the invention such that when incorporated into a microorganism host cell the protein may readily be expressed as required, if necessary by addition of suitable inducers.
The luc genes for
Photinus pyralis, Luciola mingrelica, Luciola cruciata
and
Luciola lateralis
are all known and isolatable by standard molecular biology techniques. This is also the case for
Lampris noctiluca. Photinus pyralis
luc gene is commercially available from Promega as the plasmid pGEM-luc. Thus convenient methods and sources for deriving starting material for production of DNA of the invention are (i) use of naturally occurring firefly genomic DNA and amplifying the luc gene from it using eg, PCR, (ii) pGEM and (iii) pGLf37 plasmid of Kajiyama and Nakano. Further genes encoding for proteins having luciferase activity, ie the activity of oxidising luciferin with the emission of light, will also be suitable sources for starting material for obtaining a DNA, and ultimately through gene expression, a protein of the invention.
Suitable vectors for use in manipulating wild type or other luc gene DNA in order to produce the DNA of the invention will be any vector in which the DNA can be contained within while alteration of the naturally occurring glutamate to an alternative amino acid is carried out. For chemically induced mutagenesis, eg using agents such as hydroxylamine, this is not particularly critical and many suitable vectors will occur to those skilled in the art that will allow easy manipulation of the gene before and after the mutagenic process. It may be preferred to specifically mutate the luc gene at the glutamate and thus a site directed mutagenesis operation will be required. Such operations may be most easily carried out in vectors and these will be well known to those skilled in the art.
For expression of luc genes of wild and known type, and those of the present invention suitable vectors include pKK223-3, pDR540 (available from Boehringer Mannheim) and pT7-7; the first two having the tac promoter under control of the lactose repressor allowing expression to be induced by presence of isopropyl-thiogalactoside (IPTG). pT7-7 allows control by the T7-RNA polymerase promoter and thus provides the basis for a very high level of gene expression in
E. coli
cells containing T7 RNA polymerase. Of these vectors expression is found to be highest when the luc genes are inserted into the pT7-7 vector.
Expression of luciferase from a luc gene inserted into pKK223-3 and pDR540 results in the expression of wild-type N-terminal sequence luciferase whilst expression from a luc gene inserted into pT7-7 results in synthesis of a fusion protein with extra N-terminal amino acids A-R-I-Q (SEQ ID NO: 6). The ribosome binding site and start codon of the luc gene in each of the respective vectors with the luc gene present (named constructs pPW204, pPW116 and pPW304) are shown in Table 1 of the Examples. pPW601a referred to below is derived by rem

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