U.S. patent application number 10/185151 was filed with the patent office on 2003-09-04 for mutant luciferases.
This patent application is currently assigned to Promega Corporation. Invention is credited to Wood, Keith V., Wood, Monika G..
Application Number | 20030166905 10/185151 |
Document ID | / |
Family ID | 22647117 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166905 |
Kind Code |
A1 |
Wood, Keith V. ; et
al. |
September 4, 2003 |
Mutant luciferases
Abstract
The invention provides active, non-naturally occurring mutants
of beetle luciferases and DNAs which encode such mutants. A mutant
luciferase of the invention differs from the corresponding
wild-type luciferase by producing bioluminescence with a wavelength
of peak intensity that differs by at least 1 nm from the wavelength
of peak intensity of the bioluminescence produced by the wild-type
enzyme. The mutant luciferases and DNAs of the invention are
employed in various biosensing applications.
Inventors: |
Wood, Keith V.; (Mt. Horeb,
WI) ; Wood, Monika G.; (Mt. Horeb, WI) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Promega Corporation
|
Family ID: |
22647117 |
Appl. No.: |
10/185151 |
Filed: |
June 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10185151 |
Jun 28, 2002 |
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08478205 |
Jun 7, 1995 |
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6552179 |
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08478205 |
Jun 7, 1995 |
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08177081 |
Jan 3, 1994 |
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Current U.S.
Class: |
536/23.2 ;
435/191; 435/320.1; 435/348; 435/69.1; 435/8 |
Current CPC
Class: |
C12Y 113/12007 20130101;
C12N 9/0069 20130101 |
Class at
Publication: |
536/23.2 ; 435/8;
435/69.1; 435/191; 435/348; 435/320.1 |
International
Class: |
C12Q 001/66; C07H
021/04; C12N 009/06; C12N 005/06; C12P 021/02 |
Claims
We claim:
1. An isolated DNA molecule comprising a segment having a sequence
which encodes a synthetic mutant beetle luciferase comprising an
amino acid sequence that differs from that of the corresponding
wild-type luciferase by at least one amino acid substitution, the
position of the amino acid substitution corresponding to a position
in the amino acid sequence of LucPplGR of SEQ ID NO:2 selected from
the group consisting of position 215, 224, 238, 242, 247 and 348,
wherein the mutant luciferase produces bioluminescence having a
shift in wavelength of peak intensity of at least 1 nanometer
relative to the bioluminescence produced by the wild-type
luciferase, wherein for the encoded amino acid sequence, the
corresponding wild-type luciferase is LucPplGR of SEQ ID NO:2, and
wherein the encoded synthetic mutant luciferase includes an amino
acid substitution selected from the group consisting of
LucPplGR-R.sub.215Q, -R.sub.215S, -R.sub.215Y, -R.sub.215K,
-R.sub.215C, -R.sub.215E, -R.sub.215F, -H.sub.242S, -H.sub.348A,
-V.sub.224F/L.sub.238M, and -V.sub.224F/S.sub.247Y.
2. An isolated DNA molecule according to claim 1, wherein the
encoded mutant luciferase has one amino acid substitution.
3. An isolated DNA molecule according to claim 1, wherein the
encoded mutant luciferase has two amino acid substitutions.
4. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215Q.
5. An isolated DNA molecule according to claim 1, wherein encoded
synthetic mutant luceriferase is LucPplGR-R.sub.215S.
6. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215Y.
7. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215K.
8. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215C.
9. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215E.
10. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-R.sub.215F.
11. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-H.sub.242S.
12. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is LucPplGR-H.sub.348A.
13. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is
LucPplGR-V.sub.224F/L.sub.238M.
14. An isolated DNA molecule according to claim 1, wherein the
encoded synthetic mutant luceriferase is
LucPplGR-V.sub.224F/S.sub.247Y.
15. An isolated DNA molecule comprising a segment having a sequence
which encodes a mutant beetle luciferase having an amino acid
sequence that differs from that of the corresponding wild-type
luciferase LucPplGR by at least one amino acid substitution,
wherein the encoded mutant luciferase is selected from the group
consisting of LucPplGR-R.sub.215Q, -R.sub.215S, -R.sub.215Y,
-R.sub.215K, -R.sub.215C, -R.sub.215E, -R.sub.215F, -H.sub.242S,
-H.sub.238A, -V.sub.224F/L.sub.238M, and -V.sub.224F/S.sub.247Y and
the encoded mutant luciferase produces bioluminescence having a
shift in wavelength of peak intensity of at least 1 nanometer
relative to the bioluminescence produced by the wild-type
luciferase.
Description
TECHNICAL FIELD
[0001] This invention generally relates to luciferase enzymes that
produce luminescence, like that from fireflies. More particularly,
the invention concerns mutant luciferases of beetles. The mutant
luciferases of the invention are made by genetic engineering, do
not occur in nature, and, in each case, include modifications which
cause a change in color in the luminescence that is produced. The
luciferases of the invention can be used, like their naturally
occurring counterparts, to provide luminescent signals in tests or
assays for various substances or phenomena.
BACKGROUND OF THE INVENTION
[0002] The use of reporter molecules or labels to qualitatively or
quantitatively monitor molecular events is well established. They
are found in assays for medical diagnosis, for the detection of
toxins and other substances in industrial environments, and for
basic and applied research in biology, biomedicine, and
biochemistry. Such assays include immunoassays, nucleic acid probe
hybridization assays, and assays in which a reporter enzyme or
other protein is produced by expression under control of a
particular promoter. Reporter molecules, or labels in such assay
systems, have included radioactive isotopes, fluorescent agents,
enzymes and chemiluminescent agents.
[0003] Included in the assay system employing chemiluminescence to
monitor or measure events of interest are assays which measure the
activity of a bioluminescent enzyme, luciferase.
[0004] Light-emitting systems have been known and isolated from
many luminescent organisms including bacteria, protozoa,
coelenterates, molluscs, fish, millipedes, flies, fungi, worms,
crustaceans, and beetles, particularly click beetles of genus
Pyrophorus and the fireflies of the genera Photinus, Photuris, and
Luciola. In many of these organisms, enzymes catalyze
monooxygenations and utilize the resulting free energy to excite a
molecule to a high energy state. Visible light is emitted when the
excited molecule spontaneously returns to the ground state. This
emitted light is called "bioluminescence." Hereinafter it may also
be referred to simply as "luminescence."
[0005] The limited occurrence of natural bioluminescence is an
advantage of using luciferase enzymes as reporter groups to monitor
molecular events. Because natural bioluminescence is so rare, it is
unlikely that light production from other biological processes will
obscure the activity of a luciferase introduced into a biological
system. Therefore, even in a complex environment, light detection
will provide a clear indication of luciferase activity.
[0006] Luciferases possess additional features which render them
particularly useful as reporter molecules for biosensing (using a
reporter system to reveal properties of a biological system).
Signal transduction in biosensors (sensors which comprise a
bilogical component) generally involves a two step process: signal
generation through a biological component, and signal transduction
and amplification through an electrical component. Signal
generation is typically achieved through binding or catalysis.
Conversion of these biochemical events into an electrical signal is
typically based on electrochemical or caloric detection methods,
which are limited by the free energy change of the biochemical
reactions. For most reactions this is less than the energy of
hydrolysis for two molecules of ATP, or about 70 kJ/mole. However,
the luminescence elicited by luciferases carries a much higher
energy content. Photons emitted from the reaction catalyzed by
firefly luciferase (560 nm) have 214 Kj/einstein. Furthermore, the
reaction catalyzed by luciferase is one of the most efficient
bioluminescent reactions known, having a quantum yield of nearly
0.9. This enzyme is therefore an extremely efficient transducer of
chemical energy.
[0007] Since the earliest studies, beetle luciferases, particularly
that from the common North American firefly species Photinus
pyralis, have served as paradigms for understanding of
bioluminescence. The fundamental knowledge and applications of
luciferase have been based on a single enzyme, called "firefly
luciferase," derived from Photinus pyralis. However, there are
roughly 1800 species of luminous beetles worldwide. Thus, the
luciferase of Photinus pyralis is a single example of a large and
diverse group of beetle luciferases. It is known that all beetle
luciferases catalyze a reaction of the same substrate, a
polyheterocyclic organic acid,
D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)-.DELTA..sup.2-thiazo-
line-4-carboxylic acid (hereinafter referred to as "luciferin",
unless otherwise indicated), which is converted to a high energy
molecule. It is likely that the catalyzed reaction entails the same
mechanism in each case.
[0008] The general scheme involved in the mechanism of beetle
bioluminescence appears to be one by which the production of light
takes place after the oxidative decarboxylation of the luciferin,
through interaction of the oxidized luciferin with the enzyme. The
color of the light apparently is determined by the spatial
organization of the enzyme's amino acids which interact with the
oxidized luciferin.
[0009] The luciferase-catalyzed reaction which yields
bioluminescence (hereinafter referred to simply as "the
luciferase-luciferin reaction") has been described as a two-step
process involving luciferin, adenosine triphosphate (ATP), and
molecular oxygen. In the initial reaction, the luciferin and ATP
react to form luciferyl adenylate with the elimination of inorganic
pyrophosphate, as indicated in the following reaction:
E+LH.sub.2+ATPE.multidot.LH-AMP+PP.sub.i
[0010] where E is the luciferase, LH.sub.2 is luciferin, and PPi is
pyrophosphate. The luciferyl adenylate, LH.sub.2-AMP, remains
tightly bound to the catalytic site of luciferase. When this form
of the enzyme is exposed to molecular oxygen, the enzyme-bound
luciferyl adenylate is oxidized to yield oxyluciferin (L=0) in an
electronically excited state. The excited oxidized luciferin emits
light on returning to the ground state as indicated in the
following reaction: 1
[0011] One quantum of light is emitted for each molecule of
luciferin oxidized. The electronically excited state of the
oxidized luciferin is a characteristic state of the
luciferase-luciferin reaction of a beetle luciferase; the color
(and, therefore, the energy) of the light emitted upon return of
the oxidized luciferin to the ground state is determined by the
enzyme, as evidenced by the fact that various species of beetles
having the same luciferin emit differently colored light.
[0012] Luciferases have been isolated directly from various
sources. The cDNAs encoding luciferases of various beetle species
have been reported. (See de Wet et al., Molec. Cell. Biol 7,
725-737 (1987); Masuda et al., Gene 77, 265-270 (1989); Wood et
al., Science 244, 700-702 (1989)). With the cDNA encoding a beetle
luciferase in hand, it is entirely straightforward for the skilled
to prepare large amounts of the luciferase by isolation from
bacteria (e.g., E. coli), yeast, mammalian cells in culture, or the
like, which have been transformed to express the cDNA.
Alternatively, the cDNA, under control of an appropriate promoter
and other signals for controlling expression, can be used in such a
cell to provide luciferase, and ultimately bioluminescence
catalyzed thereby, as a signal to indicate activity of the
promoter. The activity of the promoter may, in turn, reflect
another factor that is sought to be monitored, such as the
concentration of a substance that induces or represses the activity
of the promoter. Various cell-free systems, that have recently
become available to make proteins from nucleic acids encoding them,
can also be used to make beetle luciferases.
[0013] Further, the availability of cDNAS encoding beetle
luciferases and the ability to rapidly screen for cDNAS that encode
enzymes which catalyze the luciferase-luciferin reaction (see de
Wet et al., supra and Wood et al., supra) also allow the skilled to
prepare, and obtain in large amounts, other luciferases that retain
activity in catalyzing production of bioluminescence through the
luciferase-luciferin reaction. These other luciferases can also be
prepared, and the cDNAs that encode them can also be used, as
indicated in the previous paragraph. In the present disclosure, the
term "beetle luciferase" or "luciferase" means an enzyme that is
capable of catalyzing the oxidation of luciferin to yield
bioluminescence, as outlined above.
[0014] The ready availability of cDNAS encoding beetle luciferases
makes possible the use of the luciferases as reporters in assays
employed to signal, monitor or measure genetic events associated
with transcription and translation, by coupling expression of such
a cDNA, and consequently production of the enzyme, to such genetic
events.
[0015] Firefly luciferase has been widely used to detect promoter
activity in eucaryotes. Though this enzyme has also been used in
procaryotes, the utility of firefly luciferase as genetic reporter
in bacteria is not commonly recognized. As genetic reporters,
beetle luciferases are particularly useful since they are monomeric
products of a single gene. In addition, no post-translational
modifications are required for enzymatic activity, and the enzyme
contains no prosthetic groups, bound cofactors, or disulfide bonds.
Luminescence from E.coli containing the gene for firefly luciferase
can be triggered by adding the substrate luciferin to the growth
medium. Luciferin readily penetrates biological membranes and
cannot be used as a carbon or nitrogen source by E.coli. The other
substrates required for the bioluminescent reaction, oxygen and
ATP, are available within living cells. However, measurable
variations in luminescence color from luciferases would be needed
for systems which utilize two or more different luciferases as
reporters (signal geneators).
[0016] Clones of different beetle luciferases, particularly of a
single genus or species, can be utilized together in bioluminescent
reporter systems. Expression in exogenous hosts should differ
little between these luciferases because of their close sequence
similarity. Thus, in particular, the click beetle luciferases may
provide a multiple reporter system that can allow the activity of
two or more different promoters to be monitored within a single
host, or for different populations of cells to be observed
simultaneously. The ability to distinguish each of the luciferases
in a mixture, however, is limited by the width of their emissions
spectra.
[0017] One of the most spectacular examples of luminescence color
variation occurs in Pyrophorus plagiophthalamus, a large click
beetle indigenous to the Caribbean. This beetle has two sets of
light organs, a pair on the dorsal surface of the prothorax, and a
single organ in a ventral cleft of the abdomen. Four different
luciferase clones have been isolated from the ventral organ. The
luciferin-luciferase reactions catalyzed by these enzymes produces
light that ranges from green to orange.
[0018] Spectral data from the luciferase-luciferin reaction
catalyzed by these four luciferases show four overlapping peaks of
nearly even spacing, emitting green (peak intensity: 546
nanometers), yellow-green (peak intensity: 560 nanometers), yellow
(peak intensity: 578 nanometers) and orange (peak intensity: 593
nanometers) light. The respective proteins are named LucPplGR,
LucPplYG, LucPplYE and LucPplOR. Though the wavelengths of peak
intensity of the light emitted by these luciferases range over
nearly 50 nm, there is still considerable overlap among the
spectra, even those with peaks at 546 and 593 nm. Increasing the
difference in wavelength of peak intensity would thus be useful to
obtain greater measurement precision in systems using two or more
luciferases.
[0019] The amino acid sequences of the four luciferases from the
ventral organ are highly similar. Comparisons of the sequences show
them to be 95 to 99% identical.
[0020] It would be desirable to enhance the utility of beetle
luciferases for use in systems using multiple reporters to effect
mutations in luciferase-encoding cDNAs to produce mutant
luciferases which, in the luciferase-luciferin reaction, produce
light with differences between wavelengths of peak intensity that
are greater than those available using currently available
luciferases.
[0021] Beetle luciferases are particularly suited for producing
these mutant luciferases since color variation is a direct result
of changes in the amino acid sequence.
[0022] Mutant luciferases of fireflies of genus Luciola are known
in the art. Kajiyama et al., U.S. Pat. Nos. 5,219,737 and
5,229,285.
[0023] In using luciferase expression in eukaryotic cells for
biosensing, it would be desirable to reduce transport of the
luciferase to peroxisomes. Sommer et al., Mol. Biol. Cell 3,
749-759 (1992), have described mutations in the three
carboxy-terminal amino acids of P. pyralis luciferase that
significantly reduce peroxisome-targeting of the enzyme.
[0024] The sequences of cDNAs enoding various beetle luciferases,
and the amino acid sequences deduced from the cDNA sequences, are
known, as indicated in Table I.
1 Luciferase Reference LucPplGR K. Wood, Ph.D. Dissertation,
University of California, San Diego (1989), see also SEQ ID NO:1;
Wood et al., Science 244, 700-702 (1989) LucPplYG K. Wood, Ph.D.
Dissertation, University of California, San Diego (1989); Wood et
al., Science 244, 700-702 (1989) LucPplYE K. Wood, Ph.D.
Dissertation, University of California, San Diego (1989); Wood et
al., Science 244, 700-702 (1989) LucPplOR K. Wood, Ph.D.
Dissertation, University of California, San Diego (1989); Wood et
al., Science 244, 700-702 (1989) Photinus pyralis de Wet et al.,
Mol. Cell. Biol. 7, 725-737 (1987); K. Wood, Ph.D. Dissertation,
University of California, San Diego (1989); Wood et al., Science
244, 700- 702 (1989) Luciola cruciata Kajiyama et al., U.S. Pat.
No. 5,229,285; Masuda et al., U.S. Pat. No. 4,968,613 Luciola
lateralis Kajiyama et al., U.S. Pat. No. 5,229,285 Luciola
mingrelica Devine et al., Biochim. et Biophys. Acta 1173, 121-132
(1993)
[0025] The cDNA and amino acid sequences of LucPplGR, the
green-emitting luciferase of the elaterid beetle Pyrophorus
plagiophthalamus, are shown in SEQ ID NO:1 (See FIG. 1).
SUMMARY OF THE INVENTION
[0026] The present invention provides mutant luciferases of beetles
and DNAs which encode the mutant luciferases. Preferably, the
mutant luciferases produce a light of different color from that of
the corresponding wild-type luciferase and preferably this
difference in color is such that the wavelength of peak intensity
of the luminescence of the mutant differs by at least 1 nm from
that of the wild-type enzyme.
[0027] The mutant luciferases of the invention differ from the
corresponding wild-type enzymes by one or more, but typically fewer
than three, amino acid substitutions. The luciferases of the
invention may also entail changes in one or more of the three
carboxy-terminal amino acids to reduce peroxisome targeting.
[0028] In one surprising aspect of the invention, it has been
discovered that combining in a single mutant two amino acid
substitions, each of which, by itself, occasions a change in color
(shift in wavelength of peak intensity) of bioluminescence, causes
the mutant to have a shift in wavelength of peak intensity that is
greater than either shift caused by the single amino acid
substitutions.
[0029] cDNAs encoding the mutant luciferases of the invention may
be obtained straightforwardly by any standard, site-directed
mutagenesis procedure carried out with a cDNA encoding the
corresponding wild-type enzyme or another mutant. The mutant
luciferases of the invention can be made by standard procedures for
expressing the cDNAs which encode them in prokaryotic or eukaryotic
cells.
[0030] A fuller appreciation of the invention will be gained upon
examination of the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0031] In the following description and examples, process steps are
carried out and concentrations are measured at room temperature
(about 20.degree. C. to 25.degree. C.) and atmospheric pressure
unless otherwise specified.
[0032] All amino acids referred to in the specification, except the
non-enantiomorphic glycine, are L-amino acids unless specified
otherwise. An amino acid may be referred to using the one-letter or
three-letter designation, as indicated in the following Table
II.
2TABLE II Designations for Amino Acids Three-Letter One-Letter
Amino Acid Designation Designation L-alanine Ala A L-arginine Arg R
L-asparagine Asn N L-aspartic acid Asp D L-cysteine Cys C
L-glutamic acid Glu E L-glutamine Gln Q glycine Gly G L-histidine
His H L-isoleucine Ile I L-leucine Leu L L-lysine Lys K
L-methionine Met M L-phenylalanine Phe F L-proline Pro P L-serine
Ser S L-threonine Thr T L-tryptophan Trp W L-tyrosine Tyr Y
L-valine Val V
[0033] "X" means any one of the twenty amino acids listed in Table
II.
[0034] Peptide or polypeptide sequences are written and numbered
from the initiating methionine, which is numbered "1," to the
carboxy-terminal amino acid.
[0035] A substitution at a position in a polypeptide is indicated
with [designation for original amino acid].sub.[position
number][designation for replacing amino acid]. For example,
substitution of an alanine at position 100 in a polypeptide with a
glutamic acid would be indicated by Ala.sub.100Glu or A.sub.100E.
Typically, the substitution will be preceded by a designation for
the polypeptide in which the substitution occurs. For example, if
the substitution A.sub.100E occurs in an hypothetical protein
designated "Luck," the substitution would be indicated as
Luck-Ala.sub.100Glu or Luck-A.sub.100E. If there is more than one
substitution in a polypeptide, the indications of the substitutions
are separated by slashes. For example, if the hypothetical protein
"Luck" has a substitution of glutamic acid for alanine at position
100 and a substitution of asparagine for lysine at position 150,
the polypeptide with the substitutions would be indicated as
Luck-Ala.sub.100Glu/Lys.sub.150Asn or Luck-A.sub.100E/K.sub.150N.
To indicate different substitutions at a position in a polypeptide,
the designations for the substituting amino acids are separated by
commas. For example, if the hypothetical "Luck" has substitutions
of glutamic acid, glycine or lysine for alanine at position 100,
the designation would be Luck-Ala.sub.100/Glu, Gly, Lys or
Luck-A.sub.100/E, G, K.
[0036] The standard, one-letter codes "A," "C," "G," and "T" are
used herein for the nucleotides adenylate, cytidylate, guanylate,
and thymidylate, respectively. The skilled will understand that, in
DNAs, the nucleotides are 2'-deoxyribonucleotide-5'-phosphates (or,
at the 5'-end, triphosphates) while, in RNAs, the nucleotides are
ribonucleotide-5'-phosphates (or, at the 5'-end, triphosphates) and
uridylate (U) occurs in place of T. "N" means any one of the four
nucleotides.
[0037] Oligonucleotide or polynucleotide sequences are written from
the 5'-end to the 3'-end.
[0038] The term "mutant luciferase" is used herein to refer to a
luciferase which is not naturally occurring and has an amino acid
sequence that differs from those of naturally occurring
luciferases.
[0039] In one of its aspects, the present invention is a mutant
beetle luciferase which produces bioluminescence (i.e., catalyzes
the oxidation of luciferin to produce bioluminescence) which has a
shift in wavelength of peak intensity of at least 1 nm from the
wavelength of peak intensity of the bioluminescence produced by the
corresponding wild-type luciferase and has an amino acid sequence
that differs from that of the corresponding wild-type luciferase by
a substitution at one position or substitutions at two positions;
provided that, if there is a substitution at one position, the
position corresponds to a position in the amino acid sequence of
LucPplGR selected from the group consisting of position 214, 215,
223, 224, 232, 236, 237, 238, 242, 244, 245, 247, 248, 282, 283 and
348; provided further that, if there are substitutions at two
positions, at least one of the positions corresponds to a position
in the amino acid sequence of LucPplGR selected from the group
consisting of position 214, 215, 223, 224, 232, 236, 237, 238, 242,
244, 245, 247, 248, 282, 283 and 348; and provided that the mutant
optionally has a peroxisome-targeting-avoiding sequence at its
carboxy-terminus.
[0040] Exemplary mutant luciferases of the invention are those of
the group consisting of LucPplGR-R.sub.215H, -R.sub.215G,
-R.sub.215T, -R.sub.215M, -R.sub.215P, -R.sub.215A, -R.sub.215L,
-R.sub.223L, -R.sub.223Q, -R.sub.223M, -R.sub.223H, -V.sub.224I,
-V.sub.224S, -V.sub.224F, -V.sub.224Y, -V.sub.224L, -V.sub.224H,
-V.sub.224G, -V.sub.232E, -V.sub.236H, -V.sub.236W, -Y.sub.237S,
-Y.sub.237C, -L.sub.238R, -L.sub.238M, -L.sub.238Q, -L.sub.238S,
-L.sub.238D, -H.sub.242A, -F.sub.244L, -G.sub.245S, -G.sub.245E,
-S.sub.247H, -S.sub.247T, -S.sub.247Y, -S.sub.247F, -I.sub.248R,
-I.sub.248V, -I.sub.248F, -I.sub.248T, -I.sub.248S, -I.sub.248N,
-H.sub.348N, -H.sub.348Q, -H.sub.348E, -H.sub.348C,
-S.sub.247F/F.sub.246L, -S.sub.247F/I.sub.248C,
-S.sub.247F/I.sub.248T, -V.sub.224F/R.sub.215G,
-V.sub.224F/R.sub.215T, -V.sub.224F/R.sub.215V,
-V.sub.224F/R.sub.215P, -V.sub.224F/P.sub.222S,
-V.sub.224F/Q.sub.227E, -V.sub.224F/L.sub.238V,
-V.sub.224F/L.sub.238T, -V.sub.224F/S.sub.247G,
-V.sub.224F/S.sub.247H, -V.sub.224F/S.sub.247T, and
-V.sub.224F/S.sub.247F.
[0041] The following Table III shows spectral properties of these
and other exemplary mutant luciferases.
3 TABLE III Protein Spectral Properties LucPplGR- peak shift width
w.t. 545 0 72 V.sub.214S * Q * Y * K * L * G * C * E * F * P * H *
R * R.sub.215H 562 17 82 Q 567 22 81 G 576 31 82 T 576 31 84 M 582
37 83 P 588 43 91 S * Y * K * L * C * E * F * R.sub.223L 549 4 75 Q
549 4 73 R.sub.223M 549 4 75 H 551 6 75 S * Y * K * G * C * E * F *
P * V.sub.224I 546 1 75 S 556 11 70 F 561 16 84 Y 565 20 87 L 578
33 94 H 584 39 69 G 584 39 70 V.sub.232E 554 9 83 V.sub.236H 554 9
74 W 554 9 74 Y.sub.237S 553 8 73 C 554 9 74 L.sub.238R 544 -1 72 M
555 10 75 Q 557 12 76 S 559 14 73 D 568 23 76 H.sub.242A 559 14 75
H.sub.242S 561 16 74 F.sub.244L 555 10 73 G.sub.245S 558 13 75 E
574 29 79 S.sub.247H 564 19 72 Y 566 21 79 F 569 24 84 I.sub.248R
544 -1 72 V 546 1 72 F 548 3 74 T 554 9 75 S 558 13 80 N 577 32 90
H.sub.348A 592 47 67 C 593 48 66 N 597 52 67 Q 605 60 72
V.sub.214C/V.sub.224A 559 14 72 S.sub.247F/F.sub.246L 567 22 79
S.sub.247F/I.sub.248C 586 41 84 S.sub.247F/I.sub.248T 596 51 80
T.sub.233A/L.sub.238M 555 10 75 V.sub.282I/I.sub.283V 563 3 73
V.sub.224F/R.sub.215G 584 39 80 V.sub.224F/R.sub.215T 587 42 80
V.sub.224F/R.sub.215V 589 44 80 V.sub.224F/R.sub.215P 597 52 81
V.sub.224F/P.sub.222S 564 3 86 V.sub.224F/Q.sub.227E 583 38 85
V.sub.224F/L.sub.238V 575 30 85 V.sub.224F/L.sub.238M 576 31 87
V.sub.224F/S.sub.247G 581 36 84 V.sub.224F/S.sub.247H 581 36 79
V.sub.224F/S.sub.247Y 595 50 88 V.sub.224F/S.sub.247F 597 52 85
*Spectral shift (.gtoreq.2 nm) observed by eye.
[0042] "Corresponding positions" in luciferases other than LucPplGR
can be determined either from alignments at the amino acid level
that are already known in the art (see, e.g., Wood et al., Science
244, 700-702 (1989); Devine et al., Biochim. et Biophys. Acta 1173,
121-132(1993)) or by simply aligning at the amino acid level to
maximize alignment of identical or conservatively substituted
residues, and keeping in mind in particular that amino acids
195-205 in the LucPplGR sequence are very highly conserved in all
beetle luciferases and that there are no gaps for more than 300
positions after that highly conserved 11-mer in any beetle
luciferase aminio acid sequence.
[0043] A "peroxisome-targeting-avoiding sequence at its
carboxy-terminus" means (1) the three carboxy-terminal amino acids
of the corresponding wild-type luciferase are entirely missing from
the mutant; or (2) the three carboxy-terminal amino acids of the
corresponding wild-type luciferase are replaced with a sequence, of
one, two or three amino acids that, in accordance with Sommer et
al., supra, will reduce peroxisome-targeting by at least 50%. If
the three carboxy-terminal amino acids of the wild-type luciferase
are replaced by a three-amino-acid peroxisome-targeting-avoiding
sequence in the mutant, and if the sequence in the mutant is
X.sub.1X.sub.2X.sub.3, where X.sub.3 is carboxy-terminal, than
X.sub.1 is any of the twenty amino acids except A, C, G, H, N, P,
Q, T and S, X.sub.2 is any of the twenty amino acids except H, M,
N, Q, R, S and K, and X.sub.3 is any of the twenty amino acids
except I, M, Y and L. Further, any one or two, or all three, of
X.sub.1, X.sub.2, and X.sub.3 could be absent from the mutant
(i.e., no amino acid corresponding to the position). The most
preferred peroxisome-targeting-avoiding sequence is IAV, where V is
at the carboxy-terminus.
[0044] In another of its aspects, the invention entails a
combination of luciferases, in a cell (eukaryotic or prokaryotic),
a solution (free or linked as a reporter to an antibody,
antibody-fragment, nucleic acid probe, or the like), or adhererd to
a solid surface, optionally through an antibody, antibody fragment
or nucleic acid, and exposed to a solution, provided that at least
one of the luciferases is a mutant, both of the luciferases remain
active in producing bioluminescence, and the wavelengths of peak
intensities of the bioluminescence of the luciferases differ
because the amino acid sequences of the luciferases differ at at
least one of the positions corresponding to positions 214, 215,
223, 224, 232, 236, 237, 238, 242, 244, 245, 247, 248, 282, 283 and
348 in the amino acid sequence of LucPplGR, provided that one or
both of the luciferases optionally have
peroxisome-targeting-avoiding sequences.
[0045] In another of its aspects, the invention entails a DNA
molecule, which may be an eukaryotic or prokaryotic expression
vector, which comprises a segment which has a sequence which
encodes a mutant beetle luciferase of the invention.
[0046] Most preferred among the DNAs of the invention are those
with segments which encode a preferred mutant luciferase of the
invention.
[0047] From the description of the invention provided herein, the
skilled will recognize many modifications and variations of what
has been described that are within the spirit of the invention. It
is intended that such modifications and variations also be
understood as part of the invetion.
Sequence CWU 1
1
* * * * *