# Patent

## Title
Mutant luciferase

## Abstract

An isolated recombinant luciferase having luciferase activity. The recombinant luciferase has an amino acid sequence which differs from the wild-type luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photinus pennsylvanica. In the sequence of the recombinant luciferase, the amino acid residue corresponding to phenylalanine 295 in Photinus pyralis wild-type luciferase or to leucine 297 in Luciola mingrelica, Luciola cruciata or Luciola lateralis wild-type luciferases, is mutated compared to the corresponding amino acid which appears in the corresponding wild-type luciferase sequence. The recombinant luciferase has increased thermostability compared to the corresponding wild-type luciferase.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 09/763,824, filed Feb. 27, 2001, which is a national stage filing under 35 USC 371 of International Application No. PCT/GB99/03538, filed Oct. 26, 1999, which claims priority benefits to United Kingdom Patent Application No. 9823468.5, filed Oct. 28, 1998. These applications are incorporated herein by reference in their entirety.


SUMMARY
The present invention relates to novel proteins, in particular mutant luciferase enzymes having increased thermostability as compared to the corresponding wild type enzyme, to the use of these enzymes in assays and to test kits containing them.
Firefly luciferase catalyses the oxidation of luciferin in the presence of ATP, Mg2+ and molecular oxygen with the resultant production of light. This reaction has a quantum yield of about 0.88. The light emitting property has led to its use in a wide variety of luminometric assays where ATP levels are being measured. Examples of such assays include those which are based upon the described in EP-B-680515 and WO 96/02665.
Luciferase is obtainable directly from the bodies of insects, in particular beetles such as fireflies or glow-worms. Particular species from which luciferases have been obtained include the Japanese GENJI or KEIKE fireflies, Luciola cruciata and Luciola lateralis, the East European firefly Luciola mingrelica, the North American firefly Photinus pyralis and the glow-worm Lampyris noctiluca. Other species from which luciferase can be obtained are listed in Ye et al., Biochimica et Biophysica Acta, 1339 (1997) 39-52. Yet a further species is Phrixothrix (railroad-worms), as described by Viviani et al., Biochemistry, 38, (1999) 8271-8279.
However, since many of the genes encoding these enzymes have been cloned and sequenced, they may also be produced using recombinant DNA technology. Recombinant DNA sequences encoding the enzymes are used to transform microorganisms such as E. coli which then express the desired enzyme product.
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. This instability causes problems when the enzyme is used or stored at high ambient temperature, or if the assay is effected under high temperature reaction conditions, for example in order to increase reaction rate.
Mutant luciferases having increased thermostability are known from EP-A-524448 and WO95/25798. The first of these describes a mutant luciferase having a mutation at position 217 in the Japanese firefly luciferase, in particular by replacing a threonine residue with an isoleucine residue. The latter describes mutant luciferases having over 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis but in which the amino acid residue corresponding to residue 354 of Photinus pyralis or 356 of the Luciola species is mutated such that it is other than glutamate.
The applicants have found yet further mutants which can bring about increased thermostability and which may complement the mutations already known in the art.
The present invention provides a protein having luciferase activity and at least 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia nayako, Photinus pennsylanvanica or Phrixothrix, wherein in the sequence of the enzyme, at least one of
(a) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase or to residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase; 
(b) the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase or to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase; 
(c) the amino acid residue corresponding to residue 295 in Photinus pyralis luciferase or to residue 297 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase; 
(d) the amino acid residue corresponding to amino acid 14 of the Photinus pyralis luciferase or to residue 16 of Luciola mingrelica, 6 residue 17 of Luciola cruciata or Luciola lateralis; 
(e) the amino acid residue corresponding to amino acid 35 of the Photinus pyrails luciferase or to residue 37 of Luciola mingrelica 38 of Luciola cruciata or Luciola lateralis; 
(f) the amino acid residue corresponding to amino acid residue 105 of the Photinus pyralis luciferase or to residue 106 of Luciola mingrelica, 107 of Luciola cruciata or Luciola lateralis or 108 of Luciola lateralis gene;
(g) the amino acid residue corresponding to amino acid residue 234 of the Photinus pyralis luciferase or to residue 236 of Luciola mingrelica, Luciola cruciata or Luciola lateralis; 
(h) the amino acid residue corresponding to amino acid residue 420 of the Photinus pyralis luciferase or to residue 422 of Luciola mingrelica, Luciola cruciata or Luciola lateralis; 
(i) the amino acid residue corresponding to amino acid residue 310 of the Photinus pyralis luciferase or to residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis; is different to the amino acid which appears in the corresponding wild type sequence and wherein the luciferase enzyme possesses has increased thermostability as compared to an enzyme having the amino acid of the corresponding wild-type luciferase of a particular species at this position.
Preferably, the protein has luciferase activity and at least 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia nayako, or Photinus pennsylanvanica. 
In particular, the protein is a recombinant protein which has luciferase activity and substantially the sequence of a wild-type luciferase, for example of Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus (Green-Luc GR), Pyrophorus plagiophthalamus (Yellow-Green Luc YG), Pyrophorus plaglophthalamus (Yellow-Luc YE), Pyrophorus plagiophthalamus 
(Orange-Luc OR), Lampyris noctiluca, Pyrocelia nayako Photinus pennsylanvanica LY, Photinus pennsylanvanica KW, Photinus pennsylanvanica J19, or Phrixothrix green (PvGR) or red (PhRE) but which may include one or more, for example up to 100 amino acid residues, preferably no more than 50 amino acids and more preferably no more than 30 amino acids, which have been engineered to be different to that of the wild type enzyme.
In particular, bioluminescent enzymes from species that can use the substrate D-luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazole carboxylic acid) to produce light emission may form the basis of the mutant enzymes of the invention.
By way of example, where the protein has substantially the sequence of luciferase of Photinus pyralis, in accordance with the invention, at least one of
(a) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase has been changed to be other than threonine;
(b) the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase has been changed to be other than isoleucine;
(c) the amino acid residue corresponding to residue 295 in Photinus pyralis luciferase has been changed to be other than phenylalanine;
(d) the amino acid residue corresponding to amino acid 14 of the Photinus pyralis luciferase has been changed to be other than phenylalanine;
(e) the amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase has been changed to be other than leucine;
(f) the amino acid residue corresponding to amino acid residue 105 of the Photinus pyralis luciferase has been changed to be other than alanine;
(g) the amino acid residue corresponding to amino acid residue 234 of the Photinus pyralis luciferase has been changed to be other than aspartic acid;
(h) the amino acid residue corresponding to amino acid residue 420 of the Photinus pyralis luciferase has been changed to be other than serine;
(i) the amino acid residue corresponding to amino acid residue 310 of the Photinus pyralis luciferase has been changed to be other than histidine.
Where the protein has substantially the sequence of Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme, in accordance with the invention, at least one of
(a) the amino acid residue corresponding to residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is other than glycine (for Luciola mingrelica based sequences) or aparagine (for Luciola cruciata or Luciola lateralis) based sequences;

(b) the amino acid residue corresponding to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is other than serine;

(c) amino acid residue corresponding to residue 297 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is other than leucine;

(d) amino acid residue corresponding to amino acid 16 of Luciola mingrelica, or to amino acid 17 of Luciola cruciata or Luciola lateralis is other than phenylalanine;

(e) amino acid residue corresponding to residue 37 of Luciola mingrelica, or 38 of Luciola cruciata or Luciola lateralis is other than lysine; 

(f) amino acid residue corresponding to amino acid residue 106 of Luciola mingrelica, or to amino acid 107 of Luciola cruciata or Luciola lateralis or to amino acid 108 of Luciola lateralis gene is other than glycine;

(g) amino acid residue corresponding to amino acid residue 236 of Luciola mingrelica, Luciola cruciata or Luciola lateralis is other than glycine;

(h) amino acid residue corresponding to residue 422 of Luciola mingrelica, Luciola cruciata or Luciola lateralis is other than threonine;

(i) amino acid residue corresponding to amino acid residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis is other than threonine (for Luciola mingrelica based sequences) or valine (for Luciola cruciata or Luciola lateralis) based sequences.

The particular substituted amino acids in any case which give rise to enhanced thermostability can be determined by routine methods as illustrated hereinafter. In each case, different substitutions may result in enhanced thermostability. Substitution may be effected by site-directed mutagenesis of DNA encoding native or suitable mutant proteins as would be understood by the skilled person. The invention in this case is associated with the identification of the positions which are associated with thermostability.
In general however, it may be desirable to consider substituting an amino acid of different properties to the wild type amino acid. Thus hydrophilic amino acid residues may, in some cases be preferably substituted with hydrophobic amino acid residues and vice versa. Similarly, acidic amino acid residues may be substituted with basic residues.
For instance, the protein may comprise a protein having luciferase activity and at least 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme wherein in the sequence of the enzyme, at least one of
(a) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase and to residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is mutated and is other than threonine in the case of Photinus pyralis luciferase; or

(b) the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase and to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is mutated and is other than isoleucine in the case of Photinus pyralis luciferase; or

(c) amino acid residue corresponding to residue 295 in Photinus pyralis luciferase and to residue 297 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is mutated and is for example, other than phenylalanine in the case of Photinus pyralis luciferase;

and the luciferase enzyme has increased thermostability as compared to the wild-type luciferase.

The sequences of all the various luciferases show that they are highly conserved having a significant degree of similarity between them. This means that corresponding regions among the enzyme sequences are readily determinable by examination of the sequences to detect the most similar regions, although if necessary commercially available software (e.g. “Bestfit” from the University of Wisconsin Genetics Computer Group; see Devereux et al (1984) Nucleic Acid Research 12: 387-395) can be used in order to determine corresponding regions or particular amino acids between the various sequences. Alternatively or additionally, corresponding acids can be determined by reference to L. Ye et al., Biochim. Biophys Acta 1339 (1997) 39-52. The numbering system used in this reference forms the basis of the numbering system used in the present application.
With respect to the possible change of the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase, the polar amino acid threonine is suitably replaced with a non polar amino acid such as alanine, glycine, valine, lecine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine. A particularly preferred substitution for the threonine residue corresponding to residue 214 in Photinus pyralis is alanine. A more preferred substitution is cysteine. However, different polar residues such as asparagine at this position may also enhance the thermostability of the corresponding enzyme having threonine at this position.
Other amino acids which appear at this position in wild-type luciferase enzymes include glycine (Luciola mingrelica, Hotaria paroula), asparagine (Pyrophorus plagiophthalamus, GR, YC, YE and OR, Luciola cruciata, Luciola lateralis, Lampyris noctiluca, Pyrocelia nayako Photinus pennsylanvanica LY, J29) and serine (position 211 in Phrixothrix luciferase). These may advantageously be substituted with non-polar or different non-polar side chains such as alanine and cysteine.
As regards the possible change of the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase, the nonpolar amino acid isoleucine is suitably replaced with a different non polar amino acid such as alanine, glycine, valine, leucine, proline, phenylalanine, methionine, tryptophan or cysteine. Other amino acids appearing at this position in wild type sequences include serine and asparagine (as well as valine or alanine at corresponding position 229 in Phritothix green and red respectively). Suitably, these polar residues are substituted by non-polar residues such as those outlined above. A particularly preferred substitution for the residue corresponding to residue 232 in Photinus pyralis luciferase and to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is alanine, where this represents a change of amino acid over the wild-type sequence. Changes of the amino acid residue corresponding to residue 295 in Photinus pyralis luciferase and to residue 297 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase, may also affect the thermostability of the protein. (This corresponds to position 292 in Phrixothix luciferase.) In general, the amino acid at this position is a non-polar amino acid phenylalanine or leucine. These are suitably changed for different non-polar amino acids. For example, in Photinus pyralis, the non-polar amino acid phenylalanine is suitably replaced with a different non polar amino acid, such as alanine, leucine, glycine, valine, isoleucine, proline, methionine, tryptophan or cysteine. A particularly preferred substitution for the phenylalanine residue corresponding to residue 214 in Photinus pyralis luciferase is leucine.
Mutation at the amino acid residue corresponding to amino acid 14 of the Photinus pyralis luciferase or to amino acid 16 in Luciola luciferase, (13 in Phrixothrix luciferase) is also possible. This amino acid residue (which is usually phenylalanine, but may also be leucine, serine, arginine or in some instances tyrosine) is suitably changed to a different amino acid, in particular to a different nonpolar amino acid such as alanine, valine, leucine, isoleucine, proline, methionine or tryptophan, preferably alanine.
Mutation at the amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase or to amino acid residue 37 in Luciola mingrelica luciferase (corresponding to amino acid 38 in other Luciola spp. And in Phrixothrix) may also be effective. This amino acid varies amongst wild type enzymes, which may include leucine (Photinus pyralis) but also lysine, histidine, glycine, alanine, glutamine and aspartic acid at this position. Suitably the amino residue at this position is substituted with a non-polar amino acid residue or a different non-polar amino acid such as
such as alanine, valine, phenylalanine, isoleucine, proline, methionine or tryptophan. A preferred amino acid at this position is alanine, where this is different to the wild-type enzyme.
Mutations at the amino acid corresponding to position 14 of the Photinus pyralis sequence and/or mutation at the amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase are preferably not the only mutation in the enzyme.
They are suitably accompanied by others of the mutations defined above, in particular those at positions corresponding to positions 214, 395 or 232 of Photinus pyralis luciferase.
Changes of the amino acid residue corresponding to residue 105 in Photinus pyralis luciferase and to residue 106 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase, (102 in Phrixothrix) may also affect the thermostability of the protein. In general, the amino acid at this position is a non-polar amino acid alanine or glycine, or serine in Phrixothrix. These are suitably changed for different non-polar amino acids. For example, in Photinus pyralis, the non-polar amino acid alanine is suitably replaced with a different no npolar amino acid, such as phenylalanine, leucine, glycine, valine, isoleucine, proline, methionine or tryptophan. A particularly preferred substitution for the alanine residue corresponding to residue 105 in Photinus pyralis luciferase is valine.
Changes of the amino acid residue corresponding to residue 234 in Photinus pyralis luciferase and to residue 236 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase (231 in Phrixothrix), may also affect the thermostability of the protein. In general, the amino acid at this position is aspartic acid or glycine and in some cases, glutamine or threonine. These are suitably changed for non-polar or different non-polar amino acids as appropriate. For example, in Photinus pyralis, the amino acid residue is aspartic acid is suitably replaced with a non polar amino acid, such as alanine, leucine, glycine, valine, isoleucine, proline, methionine or tryptophan. A particularly preferred substitution for the phenylalanine residue corresponding to residue 234 in Photinus pyralis luciferase is glycine. Where a non-polar amino acid residue such as glycine is present at this position (for example in Luciola luciferase), this may be substituted with a different non-polar amino acid.
Changes of the amino acid residue corresponding to residue 420 in Photinus pyralis luciferase and to residue 422 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase (417 in Phrixothrix green and 418 in Phrixothrix red), may also affect the thermostability of the protein. In general, the amino acid at this position is an uncharged polar amino acid serine or threonine or glycine. These are suitably changed for different uncharged polar amino acids. For example, in Photinus pyralis, the serine may be replaced with asparagine, glutamine, threonine or tyrosine, and in particular threonine.
Changes of the amino acid residue corresponding to residue 310 in Photinus pyralis luciferase and to residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase, may also affect the thermostability of the protein. The amino acid residue at this position varies amongst the known luciferase proteins, being histidine in Photinus pyralis, Pyrocelia nayalco, Lampyris noctiluca and some forms of Photinus pennsylanvanica luciferase, threonine in Luciola mingrelica, Hotaria paroula and Phrixothix (where it is amino acid 307) luciferase, valine in Luciola cruciata and Luciola lateralis, and asparagine in some Pyrophorus plagiophthalamus luciferase. Thus, in general, the amino acid at this position is hydrophilic amino acid which may be changed for a different amino acid residue which increases thermostability of the enzyme. A particularly preferred substitution for the histidine residue corresponding to residue 310 in Photinus pyralis luciferase is arginine.
Other mutations may also be present in the enzyme. For example, in a preferred embodiment, the protein also has the amino acid at position corresponding to amino acid 354 of the Photinus pyralis luciferase (356 in Luciola luciferase and 351 in Phrixothrix) changed from glutamate, in particular to an amino acid other than glycine, proline or aspartic acid. Suitably, the amino acid at this position is tryptophan, valine, leucine, isoleucine are asparagine, but most preferably is lysine or arginine. This mutation is described in WO 95/25798.
In an alternative preferred embodiment, the protein also has the amino acid at the position corresponding to amino acid 217 in Luciola luciferase (215 in Photinus pyralis) changed to a hydrophobic amino acid in particular to isoleucine, leucine or valine as described in EP-A-052448.
The proteins may contain further mutations in the sequence provided the luciferase activity of the protein is not unduly compromised. The mutations suitably enhance the properties of the enzyme or better suit it for the intended purpose in some way. This may mean that they result in enhanced thermostability and/or colour shift properties, and/or the Km for ATP of the enzymes. Examples of mutations which give rise to colour shifts are described in WO95/18853. Mutations which affect Km values are described for example in WO 96/22376 and International Patent Application No. PCT/GB98/01026 which are incorporated herein by reference.
Proteins of the invention suitably have more than one such mutation, and preferably all three of the mutations described above.
Proteins of the invention include both wild-type and recombinant luciferase enzymes. They have at least 60% similarity to the sequences of Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis or other luciferase enzymes as discussed above in the sense that at least 60% of the amino acids present in the wild-type enzymes are present in the proteins of the invention. Such proteins can have a greater degree of similarity, in particular at least 70%, more preferably at least 80% and most preferably at least 90% to the wild-type enzymes listed above. Similar proteins of this type include allelic variants, proteins from other insect species as well as recombinantly produced enzymes.
They may be identified for example, in that they are encoded by nucleic acids which hybridise with sequences which encode wild-type enzymes under stringent hybridisation conditions, preferably high stringency conditions. Such conditions would be well understood by the person skilled in the art, and are exemplified for example in Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press). In general terms, low stringency conditions can be defined as 3×SCC at about ambient temperature to about 65° C., and high stringency conditions as 0.1×SSC at about 65° C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3×SSC is three times as strong as SSC and so on.
In particular, the similarity of a particular sequence to the sequences of the invention may be assessed using the multiple alignment method described by Lipman and Pearson, (Lipman, D. J. & Pearson, W. R. (1985) Rapid and Sensitive Protein Similarity Searches, Science, vol 227, pp 1435-1441). The “optimised” percentage score should be calculated with the following parameters for the Lipman-Pearson algorithm:ktup=1, gap penalty=4 and gap penalty length=12. The sequence for which similarity is to be assessed should be used as the “test sequence” which means that the base sequence for the comparison, such as the sequence of Photinus pyralis or any of the other sequences listed above, as recorded in Ye et al., supra., or in the case of Phrixotrix, as described in Biochemistry, 1999, 38, 8271-8279, should be entered first into the algorithm. Generally, Photinus pyralis will be used as the reference sequence.
Particular examples of proteins of the invention are wild-type luciferase sequence with the mutations as outlined above. The proteins have at least one and preferably more than one such mutation.
The invention further provides nucleic acids which encode the luciferases as described above. Suitably, the nucleic acids are based upon wild-type sequences which are well known in the art. Suitable mutation to effect the desired mutation in the amino acid sequence would be readily apparent, based upon a knowledge of the genetic code.
The nucleic acids of the invention are suitably incorporated into an expression vector such as a plasmid under the control of control elements such as promoters, enhancers, terminators etc. These vectors can then be used to transform a host cell, for example a prokaryotic or eukaryotic cell such as a plant or animal cell, but in particular a prokaryotic cell such as E. coli so that the cell expresses the desired luciferase enzyme. Culture of the thus transformed cells using conditions which are well known in the art will result in the production of the luciferase enzyme which can then be separated from the culture medium. Where the cells are plant or animal cells, plants or animals may be propagated from said cells. The protein may then be extracted from the plants, or in the case of transgenic animals, the proteins may be recovered from milk. Vectors, transformed cells, transgenic plants and animals and methods of producing enzyme by culturing these cells all form further aspects of the invention.
The Photinus pyralis T214A mutant luciferase was created by random mutagenesis as described hereinafter. It was found that the T214A single point mutation has greater thermostability than wild type luciferase.
Two new triple mutant luciferases: E354K/T214A/A215L and E354K/T214A/I232A were also prepared and these also have exhibited greater thermostability.
Particular examples of mutant enzymes of Photinus pyralis which fall within the scope of the invention include the following:
I232A/E354K
T214A/I232A/E354K
A215L/I232A/E354K
T214A/I232A/E354K/A215L
I232A/E354K/T214A/F295L
I232A/E354K/T214A F295L/F14A/L35A
I232A/E354K/T214A/F295L/F14A/L35A/A215L
A105V
T214A
T214C
T214N
T295L
I232A
F14A
L35A
D234G
S420T
H310R
or equivalents of any of these when derived from the luciferases of other species.
The mutations for the creation of the triple mutant were introduced to the luciferase gene on plasmid pET23 by site-directed mutagenesis, (PCR). The oligonucleotides added to the PCR reaction in order to effect the relevant mutations are given in the Examples below.
It has been reported previously that the effect of point mutations at the 354 and 215 positions are additive. This invention provides the possibility of combining three or more such mutations to provide still greater thermostability.
Thermostable luciferase of the invention will advantageously be employed in any bioluminescent assay which utilises the luciferase/luciferin reaction as a signalling means. There are many such assays known in the literature. The proteins may therefore be included in kits prepared with a view to performing such assays, optionally with luciferin and any other reagents required to perform the particular assay.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which:
FIGS. 1A and 1B illustrate the plasmids used in the production of mutants in accordance with the invention;
FIG. 2 shows the results of heat inactivation studies on luciferases including luciferases of the invention;
FIGS. 3A-3H show the results of thermostability experiments on various luciferase mutants;
FIG. 4 shows the results of thermostability experiments on other luciferase mutants; and
FIG. 5 shows oligonucleotides (SEQ ID NOs: 1-33) used in the preparation of mutant enzymes of the invention.



EXAMPLE 1
Identification of Thermostable Mutant Luciferase
The error-prone PCR was based on the protocol devised by Fromant et al., Analytical Biochemistry, 224, 347-353 (1995).
The dNTP mix in this reaction was:
35 mM dTTP
12.5 mM dGTP
22.5 mM dCTP
14 mM dATP
The PCR conditions were:
0.5 μl (50 ng) plasmid pPW601a J54*
5.0 μl 10×KC1 reaction buffer
1 μl each of W56 and W57+ (60 pmoles of each primer)
1 μl BIOTAQ (thermostable) DNA polymerase (5U)
2 μl dNTPs (see above)
1.76 μl MgCl2 (50 mM stock)
1 μl mNCl2 (25 mM stock) [final concentration in reaction=3.26 mM]
36.7 μl dH2O
*Plasmid pPW601aJ54 is a mutated version of pPW601a (WO 95/25798) where an NdeI site has been created within the 3 bases prior to the ATG start codon. This allows for easy cloning from pPW601a into the pET23 vector.
+Primer Sequences:







W56:


(SEQ ID NO: 34)


5′ - AAACAGGGACCCATATGGAAGACGC - 3′


 


W57:


(SEQ ID NO: 35)


5′ - AATTAACTCGAGGAATTTCGTCATCGCTGAATACAG - 3′)






Cycling parameters were:

94° C.-5 min

Then 12× cycles of: 94° C.-30s

    
    
        55° C.-30s
        72° C.-5 min
        72° C.-10 min
    
    


The PCR products were purified from the reaction mix using a Clontech ADVANTAGE PCR-pure kit. An aliquot of the purified products was then digested with the restriction enzymes NdeI and XhoI. The digested PCR products were then “cleaned up” with the ADVANTAGE kit and ligated into the vector pET23a which had been digested with the same enzymes.
Ligation Conditions:
4 μl pET23a (56 ng)
5 μl PCR products (200 ng)
3 μl 5× Gibco BRL ligase reaction buffer
1 μl Gibco BRL ligase (10U)
2 μl dH2O
The ligation was carried out overnight at 16° C.
The ligated DNAs were then purified using the ADVANTAGE kit and then electroporated into electrocompetent E. coli HB101 cells (1 mm cuvettes, 1.8 Kv).
Eleven electroporations were performed and the cells were then added to 40 ml of TY broth containing 50 μg/ml ampicillin. The cells were then grown overnight at 37° C. The entire 50 ml of culture grown overnight was used to purify plasmid DNA. This is the library.
Screening the Library
An aliquot of the plasmid library was used to electroporate E. coli BL21 DE3 cells. These cells were then plated onto LB agar containing 50 μg/ml ampicillin and grown overnight at 37° C.
The next day, colonies were picked and patched onto nylon filters on LB agar+amp plates and growth continued overnight at 37° C. The next day, filters were overlaid with a solution of luciferin—500 μM in 100 mM sodium citrate pH5.0. The patches were then viewed in a darkroom. One colony/patch was picked from 200 for further analysis.
Characterisation of the Thermostable Mutant
The E. coli clone harbouring the mutant plasmid was isolated. Plasmid DNA was prepared for ABI sequencing. The entire open reading frame encoding luciferase was sequenced using 4 different oligonucleotide primers. Sequencing revealed a single point mutation at nt 640 (A→G). Giving a codon change of ACT (T) to GCT (A) at amino acid position 214.
EXAMPLE 2
Preparation of Triple Mutant Enzyme
A mutagenic oligonucleotide was then used to create this same mutation in pMOD1 (A215L/E354K) to create a triple mutant pMOD2 (A215L/E354K/T214A). This mutation also creates a unique SacI/SstI site in pMOD1.
EXAMPLE 3
Preparation of Further Triple Mutant Enzyme
The following primers were used to create the triple mutant T214A/I232A/E354K using a standard PCR reaction and with the pET23 plasmid with the T214A mutation as template:









(SEQ ID NO: 26)



CTGATTACACCCAAGGGGGATG



E354K-sense



 



(SEQ ID NO: 27)



CATCCCCCTTGGGTGTAATCAG



E354K-antisense



 



(SEQ ID NO: 30)



GCAATCAAATCGCTCCGGATACTGC



I232A-sense



 



(SEQ ID NO: 31)



GCAGTATCCGGAGCGATTTGATTGC



I232A-antisense






EXAMPLE 4
Identification of Thermostable 295 Mutant
The F295 mutant was created using the error-prone PCR method described by Fromant et al., Analytical Biochemistry, vol 224, 347-353 (1995). The PCR conditions used were as follows:
0.5 μl (50 ng) plasmid pET23
5.0 μl 10×KCI reaction buffer
1 μl primer 1-60 pmoles of each primer
1 μl primer 2
1 μl BIOTAQ (thermostable) DNA polymerase (5U)
2 μl dNTPs, in mixture 35 mM dTTP, 12.5 mM dGTP, 22.5 mM dCTP,
14 mM dATP
1.76 μl MgCl2 (50 mM stock)
1 μl MnCl2 (25 mM stock) [final concentration in reaction=3.26 mM]
36.7 μl dH2O









Primer 1 =



(SEQ ID NO: 34)



5′ - AAACAGGGACCCATATGGAAGACGC - 3′



 



Primer 2 =



(SEQ ID NO: 35)



5′ - AATTAACTCGAGGAATTTCGTCATCGCTGAATACAG - 3′






The cycling parameters were:
94° C. for 5 min
15 cycles of: 30 s@ 94° C.


    
    
        30 s@ 55° C.
        5 Min@ 72° C.

then 10 min at 72° C.

    
    


The PCR products were purified from the reaction mix using a Clontech ADVANTAGE PCR-Pure kit. An aliquot of the purified products was then digested with the restriction enzymes Ndel and Xhol. The digested PCR products were then “cleaned up” with the ADVANTAGE kit and ligated into the vector pET23a, which had been digested with the same enzymes.
The ligation conditions were as follows:
56 ng pET23a
200 ng PCR products
3 μl 5× Gibco BRL ligase reaction buffer
1 μl Gibco BRL ligase (10U)
volume made up to 10 μl with dH2O
The ligation was carried out overnight at 16° C.
The ligated DNAs were then purified using the AdvantageTM kit and then electroporated into electrocompetent Escherichia coli DH5α cells (1 mm cuvettes, 1.8 kV). 1 ml of SOC broth was added to each electroporation and the cells allowed to recover and express antibiotic resistance genes encoded by the plasmid. Aliquots of the library were inoculated onto LB agar containing 50 μg/ml ampicillin and the bacteria were grown overnight at 37° C. Nylon filter discs were then overlaid onto the agar plates and the colonies transferred to fresh plates. The original plates were left at room temperature for the colonies to re-grow. The plates with the nylon filters were incubated at 42° C. for 2 h before plates were sprayed with 500 μM luciferin in 100 mM citrate buffer pH5.0 and viewed in a darkroom.
Three thermostable colonies were selected on the basis that they still glowed after 2 h at 42° C. Plasmid DNA was isolated from these clones and sequenced, and this revealed the F295L mutation in each case.
EXAMPLE 5
Other mutants of the invention were produced by PCR using appropriate combinations of the oligonucleotides listed above as well as the following:









(SEQ ID NO: 5)



GAAAGGCCCGGCACCAGCCTATCCTCTAGAGG



F14A-sense



 



(SEQ ID NO: 6)



CCTCTAGCGGATAGGCTGGTGCCGGGCCTTTC



F14A-antisense



 



(SEQ ID NO: 9)



GAGATACGCCGCGGTTCCTGG



L35A-sense



 



(SEQ ID NO: 10)



CCAGGAACCGCGGCGTATCTC 



L35A-antisense






EXAMPLE 6
Purification of Luciferase and Heat Inactivation Studies
Cells expressing the recombinant mutant luciferases were cultured, disrupted and extracted as described in WO 95/25798 to yield cell free extracts of luciferase.
Eppendorf tubes containing the cell free extracts were incubated generally at 40° C. unless otherwise stated. Purified preparations of wild type luciferases (for comparative purposes were incubated in thermostability buffer comprising 50 mM potassium phosphate buffer pH7.8 containing 10% saturated ammonium sulphate, 1 mM dithiothreitol and 0.2% bovine serum albumin (BSA). At set times a tube was removed and cooled in an ice/water bath prior to assay with remaining assayed activity being calculated as a percentage of the initial activity or relative bioluminesce.
The results are illustrated in FIGS. 2 and 3 hereinafter. It can be seen from FIG. 2 that luciferase mutants of the invention have improved thermostability compared with the previously known mutants.
The dramatic increase in stability over wild-type luciferase (RWT) is clear from FIG. 3.
EXAMPLE 7
Investigations into the Activity of 214 Mutants
A library of 214 mutants was prepared using site-directed mutagenesis using cassette oligos (FIG. 5) and thermostable mutants selected and tested as described in Example 1. Three particularly thermostable mutants were characterised by sequencing as described in Example 1 as T214A, T214C and T214N.
O/N cultures of E. coli XL1-Blue harbouring plasmids encoding T214, T214A, T214C and T214N were lysed using the Promega lysis buffer. 50 μl of liquid extracts were then heat inactivated at 37° C. and 40° C. over various time points. Aliquots 10 μl of heated extract were then tested in the Promega live assay buffer (100 μl).
The results are shown in the following Tables


















0
4 min
8 min
22 min
(37° C.)





rwt T214
11074
5561
2555
343
RLU


T214C
106449
92471
90515
78816
RLU


T214A
63829
52017
45864
35889
RLU


T214N
60679
49144
41736
29488
RLU












% remaining activity 37° C.

















rwt T214
100
50.2
23.1
3.1



T214C
100
86.9
85.0
74.0



T214A
100
81.5
71.8
56.2



T214N
100
81.0
68.8
48.6










The experiment was repeated at 40° C. with the 3 mutants





















0
4 min
8 min
16 min







T214C
104830
79365
72088
56863
RLU



T214A
64187
43521
28691
14547
RLU



T214N
60938
38359
25100
12835
RLU













% remaining activity 40° C.














0
4 min
8 min
16 min







T214C
100
73.7
68.8
54.2



T214A
100
67.8
44.7
22.7



T214N
100
63.0
41.2
21.1










These results indicate that T214C is significantly more thermostable than either r-wt or T214A or N. This change in properties is unexpected as it is usually expected that the more cysteine residues that are present, the worse the thermostability.
EXAMPLE 8
Investigation of Other Point Mutations
A series of other Photinus pyralis mutants with single point mutations were prepared using random error-prone PCR (FIG. 5). Following, screening and sequencing of the mutants generated, the sequencing was checked using site-directed mutagenesis followed by further sequencing. These were D234G, A105V and F295L. The thermostability of these mutants as well as recombinant wild-type Photinus pyralis luciferase was tested. Protein samples in Promega lysis buffer were incubated at 37° C. for 10 minutes and their activity assayed after 2, 5 and 10 minutes. The results, showing that each mutation produced enhanced thermostability over wild type, is shown in FIG. 4.

## Claims

1. An isolated recombinant luciferase having luciferase activity and comprising a variant of wild-type Photinus pyralis luciferase of SEQ ID NO: 37, wherein the amino acid sequence of said recombinant luciferase has no more than 50 amino acid differences as compared to the amino acid sequence of SEQ ID NO: 37, wherein the amino acid residue of the recombinant luciferase corresponding to position 295 of SEQ ID NO:37 is leucine, the amino acid residue of the recombinant luciferase corresponding to position 214 of SEQ ID NO:37 is an amino acid residue other than threonine, the amino acid residue of the recombinant luciferase corresponding to position 232 of SEQ ID NO:37 is an amino acid residue other than isoleucine, and the amino acid residue of the recombinant luciferase corresponding to position 354 of SEQ ID NO:37 is an amino acid residue other than glutamic acid, and wherein the recombinant luciferase has increased thermostability as compared to the wild-type Photinus pyralis luciferase of SEQ ID NO:37.

2. The recombinant luciferase of claim 1, further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position 14 of SEQ ID NO:37.

3. The recombinant luciferase of claim 2, wherein the amino acid residue corresponding to position 14 of SEQ ID NO:37 is alanine.

4. The recombinant luciferase of claim 1, further comprising an amino acid other than leucine at the amino acid residue corresponding to position 35 of SEQ ID NO:37.

5. The recombinant luciferase of claim 4, wherein the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine.

6. The recombinant luciferase of claim 1, wherein the amino acid at the residue corresponding to position 354 of SEQ ID NO:37 is lysine.

7. The recombinant luciferase of claim 1, further comprising an amino acid other than alanine at the amino acid residue corresponding to position 215 of SEQ ID NO:37.

8. The recombinant luciferase of claim 7, wherein the amino acid residue corresponding to position 215 of SEQ ID NO:37 is lysine.

9. The recombinant luciferase of claim 1, wherein the amino acid residue corresponding to position 214 of SEQ ID NO:37 is alanine, the amino acid residue corresponding to position 232 of SEQ ID NO:37 is alanine and the amino acid residue corresponding to position 354 of SEQ ID NO:37 is lysine.

10. The recombinant luciferase of claim 1, further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position 14 of SEQ ID NO:37 and an amino acid other than leucine at the amino acid residue corresponding to position 35 of SEQ ID NO:37.

11. The recombinant luciferase of claim 10, wherein the amino acid residue corresponding to position 14 of of SEQ ID NO:37 is alanine and the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine.

12. The recombinant luciferase of claim 10, further comprising an amino acid other than alanine at the amino acid residue corresponding to position 215 of SEQ ID NO:37.

13. The recombinant luciferase of claim 12, wherein the amino acid residue corresponding to position 215 of SEQ ID NO:37 is leucine.

14. A bioluminescent assay comprising the steps of contacting the recombinant luciferase of claim 1 with luciferin and detecting bioluminescence.

15. A kit comprising the recombinant luciferase of claim 1.