U.S. patent application number 11/179411 was filed with the patent office on 2005-12-01 for renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent and the use thereof in diagnostics, high throughput screening and novelty items.
Invention is credited to Bryan, Bruce, Szczepaniak, William, Szent-Gyorgyi, Christopher.
Application Number | 20050266491 11/179411 |
Document ID | / |
Family ID | 26885412 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266491 |
Kind Code |
A1 |
Bryan, Bruce ; et
al. |
December 1, 2005 |
Renilla reniformis fluorescent proteins, nucleic acids encoding the
fluorescent and the use thereof in diagnostics, high throughput
screening and novelty items
Abstract
Isolated and purified nucleic acids encoding green fluorescent
proteins from Renilla reniformis and the green fluorescent protein
encoded thereby are also provided. Mutants of the nucleic acid
molecules and the modified encoded proteins are also provided.
Compositions and combinations comprising the green fluorescent
proteins and/or the luciferase are further provided.
Inventors: |
Bryan, Bruce; (Pinetop,
AZ) ; Szent-Gyorgyi, Christopher; (Pittsburgh,
PA) ; Szczepaniak, William; (Burlington, VT) |
Correspondence
Address: |
Lara A. Northrop
Pietragallo, Bosick & Gordon
One Oxford Centre, 38th Floor
301 Grant Street
Pittsburgh
PA
15219
US
|
Family ID: |
26885412 |
Appl. No.: |
11/179411 |
Filed: |
July 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179411 |
Jul 12, 2005 |
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09808898 |
Mar 15, 2001 |
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60189691 |
Mar 15, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/43595 20130101;
C12N 9/0069 20130101; C12Y 113/12005 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C12Q
001/66; C07H 021/04; C07K 014/435 |
Claims
What is claimed is:
1. An isolated substantially purified nucleic acid molecule
comprising a sequence of nucleotides that encodes a protein of any
of SEQ ID NOS. 15, 16, 17, 18, 19, 20, 30 and/or 31, or a protein
encoded by a Renilla mulleri, Ptilosarcus or Gaussia having at
least 80% nucleotide sequence identity thereto.
2. The isolated substantially purified nucleic acid molecule of
claim 1, wherein the encoded protein is a green fluorescent
protein.
3. The isolated substantially purified nucleic acid molecule of
claim 1, that encodes a protein having at least 90% sequence
identity to the protein of any of SEQ ID NOS. 15, 16, 17, 18, 19,
20, 30 and/or 31.
4. The isolated substantially purified nucleic acid molecule of
claim 1, comprising a sequence of nucleotides selected from the
group consisting of: (a) the coding portion of the sequence of
nucleotides set forth in any of SEQ ID NOS. 15, 16, 17, 18, 19, 20,
30 and/or 31; (b) a sequence of nucleotides that hybridizes under
high stringency to the sequence of nucleotides of (a); and (c) a
sequence of nucleotides comprising degenerate codons of (a) or
(b).
5. The isolated substantially purified nucleic acid molecule of
claim 1, wherein the nucleic acid is DNA.
6. The isolated substantially purified nucleic acid molecule of
claim 1, wherein the nucleic acid is RNA.
7. A nucleic acid probe or primer, comprising at least 14
contiguous nucleotides selected from any of the sequences of
nucleotides of claim 1.
8. The nucleic acid probe or primer of claim 7, comprising at least
16 contiguous nucleotides selected from any of the sequences of
nucleotides of claim 1.
9. The nucleic acid probe or primer of claim 8, comprising at least
30 contiguous nucleotides selected from any of the sequences of
nucleotides of claim 1.
10. A plasmid, comprising any of the sequences of nucleotides of
claim 1.
11. A recombinant host cell, comprising the plasmid of claim
10.
12. The recombinant host cell of claim 11, wherein the cell is
selected from the group consisting of a bacterial cell, a yeast
cell, a fungal cell, a plant cell, an insect cell, an animal cell
or a human cell.
13. An isolated substantially purified Renilla mulleri or
Ptilosarcus green fluorescent protein encoded by the nucleic acid
molecule of claim 1.
14. A composition, comprising the green fluorescent protein of
claim 13 and at least one component of a bioluminescence generating
system.
15. The composition of claim 14, wherein the bioluminescence
generating system is selected from those isolated from: an insect
system, a coelenterate system, a ctenophore system, a bacterial
system, a mollusk system, a crustacea system, a fish system, an
annelid system, and an earthworm system.
16. The composition of claim 15, wherein the bioluminescence
generating system is selected from those isolated from: fireflies,
Mnemiopsis, Beroe ovata, Aequorea, Obelia, Vargula, Pelagia,
Renilla, Pholas Aristostomias, Pachystomias, Poricthys, Cypridina,
Aristostomias, such Pachystomias, Malacosteus, Gonadostomias,
Gaussia, Watensia, Halisturia, Vampire squid, Glyphus, Mycotophids,
Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostus, Sea
Pens, Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia,
cuttlefish, Sepiolina, Oplophorus, Acanthophyra, Sergestes,
Gnathophausia, Argyropelecus, Yarella, Diaphus, Gonadostomias and
Neoscopelus.
17. A reporter gene construct, comprising the nucleic acid molecule
of claim 1.
18. A combination, comprising an article of manufacture, and a
green fluorescent protein encoded by the nucleic acid molecule of
claim 1.
19. The combination of claim 18, further comprising at least one
component of a bioluminescence generating system, whereby the
combination is a novelty item.
20. The combination of claim 18, wherein the component of the
bioluminescence generating system comprises a luciferase and/or a
luciferin.
21. The combination of claim 18, wherein the article of manufacture
is selected from among toys, fountains, personal care items,
cosmetics, fairy dust, foods and beverages, textile and paper
products, toy guns, pellet guns, greeting cards, fingerpaints, foot
bags, slimy play material, clothing, bubble making toys and bubbles
therefor, balloons, bath powders, body lotions, gels, body powders,
body creams, toothpastes, mouthwashes, soaps, body paints, bubble
bath, board game toys, fishing lures, egg-shaped toys, toy
cigarettes, dolls, sparklers, magic wand toys, wrapping paper,
gelatins, icings, frostings, fairy dust, beer, ornamental
transgenic plants, wine, champagne, milk, soft drinks, ice cubes,
ice, dry ice, soaps, body paints and bubble bath.
22. An antibody or a molecule or derivative of the antibody
containing the binding domain thereof that specifically binds to
the nucleic acid molecule or a protein encoded by a Renilla mulleri
or Ptilosarcus of claim 1.
23. The antibody of claim 22 that is a monoclonal antibody.
24. A nucleic acid construct, comprising a nucleotide sequence
encoding a luciferase and a sequence of nucleotides of claim 1 that
encodes a Renilla mulleri or Ptilosarcus green fluorescent
protein.
25. The nucleic acid construct of claim 24, wherein the luciferase
is encoded by: a sequence of nucleotides set forth in SEQ ID No.
17, SEQ ID No.19, or SEQ ID No. 28; a sequence of nucleotides
encoding the amino acid sequence set forth in SEQ ID No.18, SEQ ID
No. 20 or SEQ ID No.29; and a sequence of nucleotides that
hybridizes under high stringency to the sequence of nucleotides set
forth in SEQ ID No.17, SEQ ID No. 19 or SEQ ID No.28.
26. The nucleic acid construct of claim 24, that is DNA.
27. The nucleic acid construct of claim 24, that is RNA.
28. A plasmid, comprising the nucleic acid construct of claim
24.
29. A recombinant host cell, comprising the plasmid of claim
24.
30. The recombinant host cell of claim 29, wherein the cell is
selected from the group consisting of a bacterial cell, a yeast
cell, a fungal cell, a plant cell, an insect cell, and animal cell
and a human cell.
31. An isolated substantially purified luciferase and green
fluorescent protein fusion protein, wherein the green fluorescent
protein is a Renilla mulleri or Ptilosarcus green fluorescent
protein and the fusion protein is encoded by the nucleic acid
construct of claim 24.
32. A biosensor, comprising a green fluorescent protein encoded by
the nucleic acid molecule of claim 1 and a luciferase.
33. A biosensor of claim 59, further comprising a modulator.
34. A biosensor, comprising a fusion protein of claim 31.
35. A bioluminescence resonance energy transfer (BRET) system,
comprising: (a) a GFP encoded by the nucleic molecule of claim 1;
(b) a luciferase from which the GFP can accept energy; (c) a
luciferin or other substrate of the luciferase.
36. The BRET system of claim 35, further comprising one or more
modulators.
37. The BRET system of claim 35, wherein the GFP and luciferase are
each attached to a different modulator, or each are attached to the
same modulator.
38. The BRET system of claim 35, wherein a conformation change in a
modulator causes an increase in the proximity of the luciferase and
GFP.
39. The BRET system of claim 35, wherein a conformational change in
a modulator causes a decrease in the proximity of the luciferase
and GFP.
40. The BRET system of claim 35, wherein the luciferase is Renilla
mulleri luciferase.
41. A transgenic animal or plant that expresses the nucleic acid of
claim 1.
42. The transgenic animal or plant of claim 41, selected from among
fish, worms, monkeys, rodents, goats, pigs, cows, sheep, horses,
flowering plants, ornamental plants.
43. The nucleic acid molecule of claim 1, that is optimized for
expression in plant cells, yeast cells, bacterial cells, fungal
cells, insect cells or animal cells.
44. The nucleic acid molecule of claim 43, that is optimized for
expression in human cells.
45. An isolated substantially purified nucleic acid molecule
encoding a green fluorescent protein, comprising a sequence of
nucleotides that encodes the protein of SEQ ID NO. 27 or a green
fluorescent protein encoded by a Renilla reniformis having at least
80% nucleotide sequence identity thereto that is optimized for
expression in plant cells, yeast cells, bacterial cells, fungal
cells, insect cells or animal cells.
46. The nucleic acid molecule of claim 45, that is optimized for
expression in human cells.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of allowed U.S. patent
application Ser. No. 09/808,898 filed Mar. 15, 2001. Benefit of
priority is also claimed under 35 U.S.C. .sctn.119(e) to U.S.
Provisional Application Ser. No. 60/189,691, filed Mar. 15, 2000,
to Bryan et al., entitled "RENILLA RENIFORMIS FLUORESCENT PROTEINS,
NUCLEIC ACIDS ENCODING THE FLUORESCENT PROTEINS AND THE USE THEREOF
IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY ITEMS".
[0002] This application is related to U.S. patent application Ser.
No. 09/277,716, filed Mar. 26, 1999, to Bruce Bryan and Christopher
Szent-Gyorgyi, entitled "LUCIFERASES, FLUORESCENT PROTEINS, NUCLEIC
ACIDS ENCODING THE LUCIFERASES AND FLUORESCENT PROTEINS AND THE USE
THEREOF IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY
ITEMS", now U.S. Pat. No. 6,232,107. This application is related to
International PCT Application No. WO 99/49019 to Bruce Bryan and
Prolume, LTD., entitled "LUCIFERASES, FLUORESCENT PROTEINS, NUCLEIC
ACIDS ENCODING THE LUCIFERASES AND FLUORESCENT PROTEINS AND THE USE
THEREOF IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY
ITEMS."
[0003] This application is also related to subject matter in U.S.
patent application Ser. No. 08/757,046, filed Nov. 25, 1996, to
Bruce Bryan entitled "BIOLUMINESCENT NOVELTY ITEMS", now U.S. Pat.
No. 5,876,995, issued Mar. 2, 1999, and in U.S. patent application
Ser. No. 08/597,274, filed Feb. 6, 1996, to Bruce Bryan, entitled
"BIOLUMINESCENT NOVELTY ITEMS", now U.S. Pat. No. 6,247,995. This
application is also related to U.S. patent application Ser. No.
08/908,909, filed Aug. 8, 1997, to Bruce Bryan entitled "DETECTION
AND VISUALIZATION OF NEOPLASTIC TISSUE AND OTHER TISSUES", now U.S.
Pat. No. 6,416,960. The application is also related to U.S. patent
application Ser. No. 08/990,103, filed Dec. 12, 1997, to Bruce
Bryan entitled "APPARATUS AND METHODS FOR DETECTING AND IDENTIFYING
INFECTIOUS AGENTS", now U.S. Pat. No. 6,458,547.
[0004] Where permitted, the subject matter of each of the above
noted applications and patents is herein incorporated by reference
in its entirety.
FIELD OF INVENTION
[0005] Provided herein are isolated and purified nucleic acids and
encoded fluorescent proteins from Renilla reniformis and uses
thereof.
BACKGROUND OF THE INVENTION
[0006] Luminescence is a phenomenon in which energy is specifically
channeled to a molecule to produce an excited state. Return to a
lower energy state is accompanied release of a photon (hy).
Luminescence includes fluorescence, phosphorescence,
chemiluminescence and bioluminescence. Bioluminescence is the
process by which living organisms emit light that is visible to
other organisms. Luminescence may be represented as follows:
A+B..fwdarw.X*+Y
X*.fwdarw.X+hv,
[0007] where X* is an electronically excited molecule and hy
represents light emission upon return of X* to a lower energy
state. Where the luminescence is bioluminescence, creation of the
excited state is derived from an enzyme catalyzed reaction. The
color of the emitted light in a bioluminescent (or chemiluminescent
or other luminescent) reaction is characteristic of the excited
molecule, and is independent from its source of excitation and
temperature.
[0008] An essential condition for bioluminescence is the use of
molecular oxygen, either bound or free in the presence of a
luciferase. Luciferases, are oxygenases, that act on a substrate,
luciferin, in the presence of molecular oxygen and transform the
substrate to an excited state. Upon return to a lower energy level,
energy is released in the form of light (for reviews see, e.g.,
McElroy et al. (1966) in Molecular Architecture in Cell Physiology,
Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J.,
pp. 63-80; Ward et al., Chapter 7 in Chemi- and Bioluminescence,
Burr, ed., Marcel Dekker, Inc. NY, pp.321-358; Hastings, J. W. in
(1995) Cell Physiology Source Book, N. Sperelakis (ed.), Academic
Press, pp 665-681; Luminescence, Narcosis and Life in the Deep Sea,
Johnson, Vantage Press, NY, see, esp. pp. 50-56).
[0009] Though rare overall, bioluminescence is more common in
marine organisms than in terrestrial organisms. Bioluminescence has
developed from as many as thirty evolutionarily distinct origins
and, thus, is manifested in a variety of ways so that the
biochemical and physiological mechanisms responsible for
bioluminescence in different organisms are distinct. Bioluminescent
species span many genera and include microscopic organisms, such as
bacteria (primarily marine bacteria including Vibrio species),
fungi, algae and dinoflagellates, to marine organisms, including
arthropods, mollusks, echinoderms, and chordates, and terrestrial
organisms including annelid worms and insects.
[0010] Assays Employing Bioluminescence
[0011] During the past twenty years, high-sensitivity biochemical
assays used in research and in medicine have increasingly employed
luminescence and fluorescence rather than radioisotopes. This
change has been driven partly by the increasing expense of
radioisotope disposal and partly by the need to find more rapid and
convenient assay methods. More recently, the need to perform
biochemical assays in situ in living cells and whole animals has
driven researchers toward protein-based luminescence and
fluorescence. The uses of firefly luciferase for ATP assays,
aequorin and obelin as calcium reporters, Vargula luciferase as a
neurophysiological indicator, and the Aequorea green fluorescent
protein as a protein tracer and pH indicator show the potential of
bioluminescence-based methods in research laboratories.
[0012] Bioluminescence is also beginning to directly impact
medicine and biotechnology; for example, Aequorea green fluorescent
protein (GFP) is employed to mark cells in murine model systems and
as a reporter in high throughput drug screening. Renilla luciferase
is under development for use in diagnostic platforms.
[0013] Bioluminescence Generating Systems
[0014] Bioluminescence, as well as other types of
chemiluminescence, is used for quantitative determinations of
specific substances in biology and medicine. For example,
luciferase genes have been cloned and exploited as reporter genes
in numerous assays, for many purposes. Since the different
luciferase systems have different specific requirements, they may
be used to detect and quantify a variety of substances. The
majority of commercial bioluminescence applications are based on
firefly (Photinus pyralis) luciferase. One of the first and still
widely used assays involves the use of firefly luciferase to detect
the presence of ATP. It is also used to detect and quantify other
substrates or co-factors in the reaction. Any reaction that
produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either
directly or indirectly, can be coupled to the light-emitting
reaction of bacterial luciferase.
[0015] Another luciferase system that has been used commercially
for analytical purposes is the Aequorin system. The purified
jellyfish photoprotein, aequorin, is used to detect and quantify
intracellular Ca.sup.2+ and its changes under various experimental
conditions. The Aequorin photoprotein is relatively small
(.about.20 kDa), nontoxic, and can be injected into cells in
quantities adequate to detect calcium over a large concentration
range (3.times.10.sup.-7 to 10.sup.-4 M).
[0016] Because of their analytical utility, luciferases and
substrates have been studied and well-characterized and are
commercially available (e.g., firefly luciferase is available from
Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals,
Indianapolis, Ind.; recombinantly produced firefly luciferase and
other reagents based on this gene or for use with this protein are
available from Promega Corporation, Madison, Wis.; the aequorin
photoprotein luciferase from jellyfish and luciferase from Renilla
are commercially available from Sealite Sciences, Bogart, Ga.;
coelenterazine, the naturally-occurring substrate for these
luciferases, is available from Molecular Probes, Eugene, Oreg.).
These luciferases and related reagents are used as reagents for
diagnostics, quality control, environmental testing and other such
analyses.
[0017] Because of the utility of luciferases as reagents in
analytical systems and the potential for use in high throughput
screening systems, there is a need to identify and isolate a
variety of luciferases that have improved or different spectral
properties compared to those presently available. For all these
reasons, it would be advantageous to have luciferases from a
variety of species, such as Gaussia and various Renilla species
available.
[0018] Fluorescent Proteins
[0019] Reporter genes, when co-transfected into recipient cells
with a gene of interest, provide a means to detect transfection and
other events. Among reporter genes are those that encode
fluorescent proteins. The bioluminescence generating systems
described herein are among those used as reporter genes. To
increase the sensitivity bioluminescence generating systems have
been combined with fluorescent compounds and proteins, such as
naturally fluorescent phycobiliproteins. Also of interest are the
fluorescent proteins that are present in a variety of marine
invertebrates, such as the green and blue fluorescent proteins,
particularly the green fluorescent protein (GFP) of Aequorea
victoria.
[0020] The green fluorescent proteins (GFP) constitute a class of
chromoproteins found only among certain bioluminescent
coelenterates. These accessory proteins are fluorescent and
function as the ultimate bioluminescence emitter in these organisms
by accepting energy from enzyme-bound, excited-state oxyluciferin
(e.g., see Ward et al. (1979) J. Biol. Chem. 254:781-788; Ward et
al. (1978) Photochem. Photobiol. 27:389-396; Ward et al. (1982)
Biochemistry 21:4535-4540).
[0021] The best characterized GFPs are those isolated from the
jellyfish species Aequorea, particularly Aequorea victoria (A.
victoria) and Aequorea forsk & lea (Ward et al. (1982)
Biochemistry 21:4535-4540; Prendergast et al. (1978) Biochemistry
17:3448-3453). Purified A. victoria GFP is a monomeric protein of
about 27 kDa that absorbs blue light with excitation wavelength
maximum of 395 nm, with a minor peak at 470 nm, and emits green
fluorescence with an emission wavelength of about 510 nm and a
minor peak near 540 nm (Ward et al. (1979) Photochem. Photobiol.
Rev 4:1-57). This GFP has certain limitations. The excitation
maximum of the wildtype GFP is not within the range of wavelengths
of standard fluorescein detection optics.
[0022] The detection of green fluorescence does not require any
exogenous substrates or co-factors. Instead, the high level of
fluorescence results from the intrinsic chromophore of the protein.
The chromophore includes modified amino acid residues within the
polypeptide chain. For example, fluorescent chromophore of A.
victoria GFP is encoded by the hexapeptide sequence, FSYGVQ,
encompassing amino acid residues 64-69. The chromophore is formed
by the intramolecular cyclization of the polypeptide backbone at
residues Ser65 and Gly67 and the oxidation of .alpha.-B bond of
residue Tyr66 (e.g., see Cody et al. (1993) Biochemistry
32:1212-1218; Shimomura (1978) FEBS Letters 104:220-222; Ward et
al. (1989) Photochem. Photobiol. 49:62S). The emission spectrum of
the isolated chromophore and the denatured protein at neutral pH do
not match the spectrum of the native protein, suggesting that
chromophore formation occurs post-translationally (e.g., see Cody
et al. (1993) Biochemistry 32:1212-1218).
[0023] In addition, the crystal structure of purified A. victoria
GFP has been determined (e.g., see Ormo (1996) Science
273:1392-1395). The predominant structural features of the protein
are an 11-stranded B barrel that forms a nearly perfect cylinder
wrapping around a single central .alpha.-helix, which contains the
modified p-hydroxybenzylideneimadaxolidinone chromophore. The
chromophore is centrally located within the barrel structure and is
completely shielded from exposure to bulk solvent.
[0024] DNA encoding an isotype of A. victoria GFP has been isolated
and its nucleotide sequence has been determined (e.g., see Prasher
(1992) Gene 111:229-233). The A. victoria cDNA contains a 714
nucleotide open reading frame that encodes a 238 amino acid
polypeptide of a calculated M.sub.r of 26,888 Da. Recombinantly
expressed A. victoria GFPs retain their ability to fluoresce in
vivo in a wide variety organisms, including bacteria (e.g., see
Chalfie et al. (1994) Science 263:802-805; Miller et al. (1997)
Gene 191:149-153), yeast and fungi (Fey et al. (1995) Gene
165:127-130; Straight et al. (1996) Curr. Biol. 6:1599-1608;
Cormack et al. (1997) Microbiology 143:303-311), Drosophila (e.g.,
see Wang et al. (1994) Nature 369:400-403; Plautz (1996) Gene
173:83-87), plants (Heinlein et al. (1995); Casper et al. (1996)
Gene 173:69-73), fish (Amsterdam et al. (1995)), and mammals (Ikawa
et al. (1995). Aequorea GFP vectors and isolated Aequorea GFP
proteins have been used as markers for measuring gene expression,
cell migration and localization, microtubule formation and assembly
of functional ion channels (e.g., see Terry et al. (1995) Biochem.
Biophys. Res. Commun. 217:21-27; Kain et al. (1995) Biotechniques
19:650-655). The A. victoria GFP, however, is not ideal for use in
analytical and diagnostic processes. Consequently GFP mutants have
been selected with the hope of identifying mutants that have single
excitation spectral peaks shifted to the red.
[0025] In fact a stated purpose in constructing such mutants has
been to attempt to make the A. victoria GFP more like the GFP from
Renilla, but which has properties that make it far more ideal for
use as an analytical tool. For many practical applications, the
spectrum of Renilla GFP is preferable to that of the Aequorea GFP,
because wavelength discrimination between different fluorophores
and detection of resonance energy transfer are easier if the
component spectra are tall and narrow rather than low and broad
(see, U.S. Pat. No. 5,625,048). Furthermore, the longer wavelength
excitation peak (475 nm) of Renilla GFP is almost ideal for
fluorescein filter sets and is resistant to photobleaching, but has
lower amplitude than the shorter wavelength peak at 395 nm, which
is more susceptible to photobleaching (Chalfie et al. (1994)
Science 263:802-805).
[0026] There exists a phylogenetically diverse and largely
unexplored repertoire of bioluminescent proteins that are a
reservoir for future development. For these reasons, it would be
desirable to have a variety of new luciferases and fluorescent
proteins, particularly, Renilla reniformis GFP available rather
than use muteins of A. victoria GFP. Published International PCT
application No. WO 99/49019 (see, also, allowed U.S. application
Ser. No. 09/277,716) provides a variety of GFPs including those
from Renilla species. It remains desirable to have a variety of
GFPs and luciferases available in order to optimize systems for
particular applications and to improve upon existing methods.
Therefore, it is an object herein to provide isolated nucleic acid
molecules encoding Renilla reniformis GFP and the protein encoded
thereby. It is also an object herein to provide bioluminescence
generating systems that include the luciferases, luciferins, and
also include Renilla reniformis GFP.
SUMMARY OF THE INVENTION
[0027] Isolated nucleic acid molecules that encode Renilla
reniformis fluorescent proteins are provided. Nucleic acid probes
and primers derived therefrom are also provided. Functionally
equivalent nucleic acids, such as those that hybridize under
conditions of high stringency to the disclosed molecules and those
that have high sequence identity, are also contemplated. Nucleic
acid molecules and the encoded proteins are set forth in SEQ ID
NOs. 23-27, an exemplary mutein is set forth in SEQ ID NO. 33. Also
contemplated are nucleic acid molecules that encode the protein set
forth in SEQ ID NO. 27.
[0028] Host cells, including bacterial, yeast and mammalian host
cells, and plasmids for expression of the nucleic acids encoding
the Renilla reniformis green fluorescent protein (GFP), are also
provided. Combinations of luciferases and the Renilla reniformis
GFP are also provided.
[0029] The genes can be modified by substitution of codons
optimized for expression in selected host cells or hosts, such as
humans and other mammals, or can be mutagenized to alter the
emission properties. Mutations that alter spectral properties are
also contemplated.
[0030] Such mutations may be identified by substituting each codon
with one encoding another amino acid, such as alanine, and
determining the effect on the spectral properties of the resulting
protein. Particular regions of interest are those in which
corresponding the sites mutated in other GFPs, such Aequora to
produce proteins with altered spectral properties are altered.
[0031] The Renilla reniformis GFP may be used in combination with
nucleic acids encoding luciferases, such as those known to those of
skill in the art and those that are described in copending allowed
U.S. application Ser. No. 09/277,716 (see, also, Published
International PCT application No. WO 99/49019).
[0032] Compositions containing the Renilla reniformis GFP or the
Renilla reniformis GFP and luciferase combination are provided. The
compositions can take any of a number of forms, depending on the
intended method of use therefor. In certain embodiments, for
example, the compositions contain a Gaussia luciferase, Gaussia
luciferase peptide or Gaussia luciferase fusion protein, formulated
for use in luminescent novelty items, immunoassays, donors in FET
(fluorescent energy transfer) assays, FRET (fluorescent resonance
energy transfer) assays, HTRF (homogeneous time-resolved
fluorescence) assays or used in conjunction with multi-well assay
devices containing integrated photodetectors, such as those
described herein.
[0033] The bioluminescence-generating system includes, in addition
to the luciferase, a Renilla reniformis GFP or mutated form
thereof. These compositions can be used in a variety of methods and
systems, such as those included in conjunction with diagnostic
systems for the in vivo detection of neoplastic tissues and other
tissues, such as those methods described herein.
[0034] Combinations of the Renilla reniformis GFP with articles of
manufacture to produce novelty items are provided. These novelty
items are designed for entertainment, recreation and amusement, and
include, but are not limited to: toys, particularly squirt guns,
toy cigarettes, toy "Halloween" eggs, footbags and board/card
games; finger paints and other paints, slimy play material;
textiles, particularly clothing, such as shirts, hats and sports
gear suits, threads and yarns; bubbles in bubble making toys and
other toys that produce bubbles; balloons; figurines; personal
items, such as cosmetics, bath powders, body lotions, gels, powders
and creams, nail polishes, make-up, toothpastes and other
dentifrices, soaps, body paints, and bubble bath; items such as
inks, paper; foods, such as gelatins, icings and frostings; fish
food containing luciferins and transgenic fish, particularly
transgenic fish that express a luciferase; plant food containing a
luciferin or luciferase, preferably a luciferin for use with
transgenic plants that express luciferase; and beverages, such as
beer, wine, champagne, soft drinks, and ice cubes and ice in other
configurations; fountains, including liquid "fireworks" and other
such jets or sprays or aerosols of compositions that are solutions,
mixtures, suspensions, powders, pastes, particles or other suitable
form. The combinations optionally include a bioluminescence
generating system. The bioluminescence generating systems can be
provided as two compositions: a first composition containing a
luciferase and a second composition containing one or more
additional components of a bioluminescence generating system.
[0035] Any article of manufacture that can be combined with a
bioluminescence-generating system as provided herein and thereby
provide entertainment, recreation and/or amusement, including use
of the items for recreation or to attract attention, such as for
advertising goods and/or services that are associated with a logo
or trademark is contemplated herein. Such uses may be in addition
to or in conjunction with or in place of the ordinary or normal use
of such items. As a result of the combination, the items glow or
produce, such as in the case of squirt guns and fountains, a
glowing fluid or spray of liquid or particles. The novelty in the
novelty item derives from its bioluminescence.
[0036] GFPS
[0037] Isolated nucleic acids that encode GFP from Renilla
reniformis are provided herein. Also provided are isolated and
purified nucleic acids that encode a component of the
bioluminescence generating system and the green fluorescent protein
(GFP) (see SEQ ID NOs. 23-27). In particular, nucleic acid
molecules that encode Renilla reniformis green fluorescent protein
(GFPs) and nucleic acid probes and primers derived therefrom are
provided. Nucleic acid molecules encoding Renilla reniformis GFP
are provided (see SEQ ID NOs. 23-26).
[0038] Nucleic acid probes and primers containing 14, 16, 30, 100
or more contiguous nucleotides from any of SEQ ID NOs. 23-26 are
provided. Nucleic acid probes can be labeled, if needed, for
detection, containing at least about 14, preferably at least about
16, or, if desired, 20 or 30 or more, contiguous nucleotides of
sequence of nucleotides encoding the Renilla reniformis GFP.
[0039] Methods using the probes for the isolation and cloning of
GFP-encoding DNA in Renilla reniformis are also provided. Vectors
containing DNA encoding the Renilla reniformis GFP are provided. In
particular, expression vectors that contain DNA encoding a Renilla
reniformis or in operational association with a promoter element
that allows for the constitutive or inducible expression of Renilla
reniformis are provided.
[0040] The vectors are capable of expressing the Renilla reniformis
GFP in a wide variety of host cells. Vectors for producing chimeric
Renilla reniformis GFP/luciferase fusion proteins and/or
polycistronic mRNA containing a promoter element and a multiple
cloning site located upstream or downstream of DNA encoding Renilla
reniformis GFP are also provided.
[0041] Recombinant cells containing heterologous nucleic acid
encoding a Renilla reniformis GFP are also provided. Purified
Renilla reniformis GFP peptides and compositions containing the
Renilla GFPs and GFP peptides alone or in combination with at least
one component of a bioluminescence-generating system, such as a
Renilla luciferase, are provided. The Renilla GFP and GFP peptide
compositions can be used, for example, to provide fluorescent
illumination of novelty items or used in methods of detecting and
visualizing neoplastic tissue and other tissues, detecting
infectious agents using immunoassays, such homogenous immunoassays
and in vitro fluorescent-based screening assays using multi-well
assay devices, or provided in kits for carrying out any of the
above-described methods. In particular, these proteins may be used
in FP (fluorescence polarization) assays, FET (fluorescent energy
transfer) assays, FRET (fluorescent resonance energy transfer)
assays and HTRF (homogeneous time-resolved fluorescence) assays and
also in the BRET assays and sensors provided herein.
[0042] Non-radioactive energy transfer reactions, such as FET or
FRET, FP and HTRF assays, are homogeneous luminescence assays based
on energy transfer and are carried out between a donor luminescent
label and an acceptor label (see, e.g., Cardullo et al. (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:8790-8794; Peerce et al. (1986)
Proc. Natl. Acad. Sci. U.S.A. 83:8092-8096; U.S. Pat. No.
4,777,128; U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S.
Pat. No. 5,279,943; and International PCT Application No. WO
92/01225). Non-radioactive energy transfer reactions using GFPs
have been developed (see, International PCT application Nos. WO
98/02571 and WO 97/28261). Non-radioactive energy transfer
reactions using GFPs and luciferases, such as a luciferase and its
cognate GFP (or multimers thereof), such as in a fusion protein,
are contemplated herein.
[0043] Nucleic acids that exhibit substantial sequence identity
with the nucleic acids provided herein are also contemplated. These
are nucleic acids that can be produced by substituting codons that
encode conservative amino acids and also nucleic acids that exhibit
at least about 80%, preferably 90 or 95% sequence identity.
Sequence identity refers to identity as determined using standard
programs with default gap penalties and other defaults as provided
by the manufacturer thereof.
[0044] The nucleic acids provide an opportunity to produce
luciferases and GFPs, which have advantageous application in all
areas in which luciferase/luciferins and GFPs have application. The
nucleic acids can be used to obtain and produce GFPs and GFPs from
other, particularly Renilla, species using the probes described
herein that correspond to conserved regions. These GFPs have
advantageous application in all areas in which GFPs and/or
luciferase/luciferins have application. For example, the GFPs
provide a means to amplify the output signal of bioluminescence
generating systems. Renilla GFP has a single excitation absorbance
peak in blue light (and around 498 nm) and a predominantly single
emission peak around 510 nm (with a small shoulder near 540). This
spectrum provides a means for it to absorb blue light and
efficiently convert it to green light. This results in an
amplification of the output. When used in conjunction with a
bioluminescence generating system that yields blue light, such as
Aequorea or Renilla or Vargula (Cypridina), the output signal for
any application, including diagnostic applications, is amplified.
In addition, this green light can serve as an energy donor in
fluorescence-based assays, such as fluorescence polarization
assays, FET (fluorescent energy transfer) assays, FRET (fluorescent
resonance energy transfer) assays and HTRF (homogeneous
time-resolved fluorescence) assays. Particular assays, herein
referred to as BRET (bioluminescence resonance energy transfer
assays in which energy is transferred from a bioluminescence
reaction of a luciferase to a fluorescent protein), are
provided.
[0045] Non-radioactive energy transfer reactions, such as FET or
FRET, FP and HTRF assays, are homogeneous luminescence assays based
on energy transfer that are carried out between a donor luminescent
label and an acceptor label (see, e.g., Cardullo et al. (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:8790-8794; Peerce et al. (1986)
Proc. Natl. Acad. Sc. U.S.A. 83:8092-8096; U.S. Pat. No. 4,777,128;
U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat. No.
5,279,943; and International PCT Application No. WO 92/01225).
Non-radioactive energy transfer reactions using GFPs have been
developed (see, International PCT application Nos. WO 98/02571 and
WO 97/28261).
[0046] Mutagenesis of the GFPs is contemplated herein, particularly
mutagenesis that results in modified GFPs that have red-shifted
excitation and emission spectra. The resulting systems have higher
output compared to the unmutagenized forms. These GFPs may be
selected by random mutagenesis and selection for GFPs with altered
spectra or by selected mutagenesis of the chromophore region of the
GFP.
[0047] The DNA may be introduced as a linear DNA molecule
(fragment) or may be included in an express ion vector for stable
or transient expression of the encoding DNA. In certain
embodiments, the cells that contain DNA or RNA encoding a Renilla
GFP also express the recombinant Renilla GFP or polypeptide. It is
preferred that the cells are selected to express functional GFPs
that retain the ability to fluorescence and that are not toxic to
the host cell. In some embodiments, cells may also include
heterologous nucleic acid encoding a component of a
bioluminescence-generating system, preferably a photoprotein or
luciferase. In preferred embodiments, the nucleic acid encoding the
bioluminescence-generating system component is isolated from the
species Aequorea, Vargula, Pleuromamma, Ptilosarcus or Renilla. In
more preferred embodiments, the bioluminescence-generating system
component is a Renilla reniformis luciferase or mulleri including
the amino acid sequence set forth in SEQ ID NO. 18 or the
Pleuromamma luciferase set forth in SEQ ID NO. 28, or the Gaussia
luciferase set forth in SEQ ID NO. 19.
[0048] The GFPs provided herein may be used in combination with any
suitable bioluminescence generating system, but is preferably used
in combination with a Renilla or Aequorea, Pleuromamma or Gaussia
luciferase.
[0049] Purified Renilla GFPs, particularly purified Renilla
reniformis GFP peptides are provided. Presently preferred Renilla
GFP for use in the compositions herein is Renilla reniformis GFP
including the sequence of amino acids set forth above and in the
Sequence Listing.
[0050] Fusions of the nucleic acid, particularly DNA, encoding
Renilla GFP with DNA encoding a luciferase are also provided
herein.
[0051] The cells that express functional luciferase and/or GFP,
which may be used alone or in conjunction with a
bioluminescence-generating system, in cell-based assays and
screening methods, such as those described herein.
[0052] Presently preferred host cells for expressing GFP and
luciferase are bacteria, yeasts, fungi, plant cells, insect cells
and animal cells.
[0053] The luciferases and GFPs or cells that express them also may
be used in methods of screening for bacterial contamination and
methods of screening for metal contaminants. To screen for
bacterial contamination, bacterial cells that express the
luciferase and/or GFP are put in autoclaves or in other areas in
which testing is contemplated. After treatment or use of the area,
the area is tested for the presence of glowing bacteria. Presence
of such bacteria is indicative of a failure to eradicate other
bacteria. Screening for heavy metals and other environmental
contaminants can also be performed with cells that contain the
nucleic dependent upon the particular heavy metal or
contaminant.
[0054] The systems and cells provided herein can be used for high
throughout screening protocols, intracellular assays, medical
diagnostic assays, environmental testing, such as tracing bacteria
in water supplies, in conjunction with enzymes for detecting heavy
metals, in spores for testing autoclaves in hospital, foods and
industrial autoclaves. Non-pathogenic bacteria containing the
systems can be included in feed to animals to detect bacterial
contamination in animal products and in meats.
[0055] Compositions containing a Renilla GFP are provided. The
compositions can take any of a number of forms, depending on the
intended method of use therefor. In certain embodiments, for
example, the compositions contain a Renilla GFP or GFP peptide,
preferably Renilla mulleri GFP or Renilla reniformis GFP peptide,
formulated for use in luminescent novelty items, immunoassays, FET
(fluorescent energy transfer) assays, FRET (fluorescent resonance
energy transfer) assays, HTRF (homogeneous time-resolved
fluorescence) assays or used in conjunction with multi-well assay
devices containing integrated photodetectors, such as those
described herein. In other instances, the GFPs are used in
beverages, foods or cosmetics.
[0056] Compositions that contain a Renilla reniformis GFP or GFP
peptide and at least one component of a bioluminescence-generating
system, preferably a luciferase, luciferin or a luciferase and a
luciferin, are provided. In preferred embodiments, the
luciferase/luciferin bioluminescence-generating system is selected
from those isolated from: an insect system, a coelenterate system,
a ctenophore system, a bacterial system, a mollusk system, a
crustacea system, a fish system, an annelid system, and an
earthworm system. Bioluminescence-generating systems include those
isolated from Renilla, Aequorea, and Vargula, Gaussia and
Pleuromamma.
[0057] Combinations containing a first composition containing a
Renilla reniformis GFP or Ptilosarcus GFP or mixtures thereof and a
second composition containing a bioluminescence-generating system
for use with inanimate articles of manufacture to produce novelty
items are provided. These novelty items, which are articles of
manufacture, are designed for entertainment, recreation and
amusement, and include, but are not limited to: toys, particularly
squirt guns, toy cigarettes, toy "Halloween" eggs, footbags and
board/card games; finger paints and other paints, slimy play
material; textiles, particularly clothing, such as shirts, hats and
sports gear suits, threads and yarns; bubbles in bubble making toys
and other toys that produce bubbles; balloons; figurines; personal
items, such as bath powders, body lotions, gels, powders and
creams, nail polishes, cosmetic including make-up, toothpastes and
other dentifrices, soaps, cosmetics, body paints, and bubble bath,
bubbles made from non-detergent sources, particularly proteins such
as albumin and other non-toxic proteins; in fishing lures and
glowing transgenic worms, particularly crosslinked polyacrylamide
containing a fluorescent protein and/or components of a
bioluminescence generating system, which glow upon contact with
water; items such as inks, paper; foods, such as gelatins, icings
and frostings; fish food containing luciferins and transgenic
animals, such as transgenic fish, worms, monkeys, rodents,
ungulates, ovine, ruminants and others, that express a luciferase
and/or Renilla reniformis GFP; transgenic worms that express
Renilla reniformis GFP and are used as lures; plant food containing
a luciferin or luciferase, preferably a luciferin for use with
transgenic plants that express luciferase and Renilla reniformis
GFP, transgenic plants that express Renilla reniformis GFP,
particularly ornamental plants, such as orchids, roses, and other
plants with decorative flowers; transgenic plants and animals in
which the Renilla reniformis GFP is a marker for tracking
introduction of other genes; and beverages, such as beer, wine,
champagne, soft drinks, milk and ice cubes and ice in other
configurations containing Renilla reniformis GFP; fountains,
including liquid "fireworks" and other such jets or sprays or
aerosols of compositions that are solutions, mixtures, suspensions,
powders, pastes, particles or other suitable forms.
[0058] Any article of manufacture that can be combined with a
bioluminescence-generating system and Renilla reniformis GFP or
with just a Renilla reniformis GFP, as provided herein, that
thereby provides entertainment, recreation and/or amusement,
including use of the items for recreation or to attract attention,
such as for advertising goods and/or services that are associated
with a logo or trademark is contemplated herein. Such uses may be
in addition to or in conjunction with or in place of the ordinary
or normal use of such items. As a result of the combination, the
items glow or produce, such as in the case of squirt guns and
fountains, a glowing fluid or spray of liquid or particles.
[0059] Methods for diagnosis and visualization of tissues in vivo
or in situ using compositions containing a Renilla reniformis GFP
and/or a Renilla reniformis or mulleri luciferase or others of the
luciferases and/or GFPs provided herein are provided. For example,
the Renilla reniformis GFP protein can be used in conjunction with
diagnostic systems that rely on bioluminescence for visualizing
tissues in situ. The systems are particularly useful for
visualizing and detecting neoplastic tissue and specialty tissue,
such as during non-invasive and invasive procedures. The systems
include compositions containing conjugates that include a tissue
specific, particularly a tumor-specific, targeting agent linked to
a targeted agent, a Renilla reniformis GFP, a luciferase or
luciferin. The systems also include a second composition that
contains the remaining components of a bioluminescence generating
reaction and/or the Renilla reniformis GFP. In some embodiments,
all components, except for activators, which are provided in situ
or are present in the body or tissue, are included in a single
composition.
[0060] Methods for diagnosis and visualization of tissues in vivo
or in situ using compositions containing a Gaussia luciferase are
provided. For example, the Gaussia luciferase or Gaussia luciferase
peptide can be used in conjunction with diagnostic systems that
rely on bioluminescence for visualizing tissues in situ. The
systems are particularly useful for visualizing and detecting
neoplastic tissue and specialty tissue, such as during non-invasive
and invasive procedures. The systems include compositions
containing conjugates that include a tissue specific, particularly
a tumor-specific, targeting agent linked to a targeted agent, a
Gaussia luciferase, a GFP or luciferin. The systems also include a
second composition that contains the remaining components of a
bioluminescence generating reaction and/or the Gaussia luciferase.
In some embodiments, all components, except for activators, which
are provided in situ or are present in the body or tissue, are
included in a single composition.
[0061] In particular, the diagnostic systems include two
compositions. A first composition that contains conjugates that, in
preferred embodiments, include antibodies directed against tumor
antigens conjugated to a component of the bioluminescence
generating reaction, a luciferase or luciferin, preferably a
luciferase are provided. In certain embodiments, conjugates
containing tumor-specific targeting agents are linked to
luciferases or luciferins. In other embodiments, tumor-specific
targeting agents are linked to microcarriers that are coupled with,
preferably more than one of the bioluminescence generating
components, preferably more than one luciferase molecule.
[0062] The second composition contains the remaining components of
a bioluminescence generating system, typically the luciferin or
luciferase substrate. In some embodiments, these components,
particularly the luciferin are linked to a protein, such as a serum
albumin, or other protein carrier. The carrier and time release
formulations permit systemically administered components to travel
to the targeted tissue without interaction with blood cell
components, such as hemoglobin that deactivates the luciferin or
luciferase.
[0063] Methods for diagnosing diseases, particularly infectious
diseases, using chip methodology (see, e.g., copending U.S.
application Ser. No. 08/990,103) a luciferase/luciferin
bioluminescence-generating system and a Renilla reniformis GFP are
provided. In particular, the chip includes an integrated
photodetector that detects the photons emitted by the
bioluminescence-generating system, particularly using luciferase
encoded by the nucleic acids provided herein and/or Renilla
reniformis GFP.
[0064] In one embodiment, the chip is made using an integrated
circuit with an array, such as an X-Y array, of photodetectors. The
surface of circuit is treated to render it inert to conditions of
the diagnostic assays for which the chip is intended, and is
adapted, such as by derivatization for linking molecules, such as
antibodies. A selected antibody or panel of antibodies, such as an
antibody specific for a bacterial antigen, is affixed to the
surface of the chip above each photodetector. After contacting the
chip with a test sample, the chip is contacted with a second
antibody linked to a Renilla GFP, a chimeric antibody-Renilla GFP
fusion protein or an antibody linked to a component of a
bioluminescence generating system, such as a luciferase or
luciferin, that are specific for the antigen. The remaining
components of the bioluminescence generating reaction are added,
and, if any of the antibodies linked to a component of a
bioluminescence generating system are present on the chip, light
will be generated and detected by the adjacent photodetector. The
photodetector is operatively linked to a computer, which is
programmed with information identifying the linked antibodies,
records the event, and thereby identifies antigens present in the
test sample.
[0065] Methods for generating chimeric GFP fusion proteins are
provided. The methods include linking DNA encoding a gene of
interest, or portion thereof, to DNA encoding a GFP coding region
in the same translational reading frame. The encoded-protein of
interest may be linked in-frame to the amino- or carboxyl-terminus
of the GFP. The DNA encoding the chimeric protein is then linked in
operable association with a promoter element of a suitable
expression vector. Alternatively, the promoter element can be
obtained directly from the targeted gene of interest and the
promoter-containing fragment linked upstream of the GFP coding
sequence to produce chimeric GFP proteins or to produce
polycistronic mRNAs that encode the Renilla reniformis GFP and a
luciferase, preferably a Renilla luciferase, more preferably
Renilla reniformis luciferase.
[0066] Methods for identifying compounds using recombinant cells
that express heterologous DNA encoding a Renilla reniformis GFP
under the control of a promoter element of a gene of interest are
provided. The recombinant cells can be used to identify compounds
or ligands that modulate the level of transcription from the
promoter of interest by measuring Renilla reniformis GFP-mediated
fluorescence. Recombinant cells expressing the chimeric Renilla
reniformis GFP or polycistronic mRNA encoding Renilla reniform is
and a lucifierase, may also be used for monitoring gene expression
or protein trafficking, or determining the cellular localization of
the target protein by identifying localized regions of GFP-mediated
fluorescence within the recombinant cell.
[0067] Other assays using the GFPs and/or luciferases are
contemplated herein. Any assay or diagnostic method known used by
those of skill in the art that employ Aequora GFPs and/or other
luciferases are contemplated herein.
[0068] Kits containing the GFPs for use in the methods, including
those described herein, are provided. In one embodiment, the kits
containing an article of manufacture and appropriate reagents for
generating bioluminescence are provided. The kits containing such
soap compositions, with preferably a moderate pH (between 5 and 8)
and bioluminescence generating reagents, including luciferase and
luciferin and the GFP are provided herein. These kits, for example,
can be used with a bubble-blowing or producing toy. These kits can
also include a reloading or charging cartridge or can be used in
connection with a food.
[0069] In another embodiment, the kits are used for detecting and
visualizing neoplastic tissue and other tissues and include a first
composition that contains the GFP and at least one component of a
bioluminescence generating system, and a second that contains the
activating composition, which contains the remaining components of
the bioluminescence generating system and any necessary activating
agents.
[0070] Thus, these kits will typically include two compositions, a
first composition containing the GFP formulated for systemic
administration (or in some embodiments local or topical
application), and a second composition containing the components or
remaining components of a bioluminescence generating system,
formulated for systemic, topical or local administration depending
upon the application. Instructions for administration will be
included.
[0071] In other embodiments, the kits are used for detecting and
identifying diseases, particularly infectious diseases, using
multi-well assay devices and include a multi-well assay device
containing a plurality of wells, each having an integrated
photodetector, to which an antibody or panel of antibodies specific
for one or more infectious agents are attached, and composition
containing a secondary antibody, such as an antibody specific for
the infectious agent that is linked to a Renilla reniformis GFP
protein, a chimeric antibody-Renilla reniformis GFP fusion protein
or F(Ab).sub.2 antibody fragment-Renilla reniformis GFP fusion
protein. A second composition contains a bioluminescence generating
system that emits a wavelength of light within the excitation range
of the Renilla mulleri GFP, such as species of Renilla or Aequorea,
for exciting the Renilla reniformis, which produces light that is
detected by the photodetector of the device to indicate the
presence of the agent.
[0072] As noted above, fusions of nucleic acid encoding the
luciferases and or GFPs provided herein with other luciferases and
GFPs are provided. Of particular interest are fusions that encode
pairs of luciferases and GFPs, such as a Renilla luciferase and a
Renilla GFP (or a homodimer or other multiple of a Renilla GFP).
The luciferase and GFP bind and in the presence of a luciferin will
produced fluorescence that is red shifted compared to the
luciferase in the absence of the GFP. This fusion or fusions in
which the GFP and luciferase are linked via a target, such as a
peptide, can be used as a tool to assess anything that interacts
with the linker.
[0073] Muteins of the GFPs and luciferases are provided. Of
particular interest are muteins, such as temperature sensitive
muteins, of the GFP and luciferases that alter their interaction,
such as mutations in the Renilla luciferase and Renilla GFP that
alters their interaction at a critical temperature.
[0074] Antibodies, polyclonal and monoclonal antibodies that
specifically bind to any of the proteins encoded by the nucleic
acids provided herein are also provided. These antibodies,
monoclonal or polyclonal, can be prepared employing standard
techniques, known to those of skill in the art. In particular,
immunoglobulins or antibodies obtained from the serum of an animal
immunized with a substantially pure preparation of a luciferase or
GFP provided herein or an or epitope-containing fragment thereof
are provided. Monoclonal antibodies are also provided. The
immunoglobulins that are produced have, among other properties, the
ability to specifically and preferentially bind to and/or cause the
immunoprecipitation of a GFP or luciferase, particularly a Renilla
or Ptilosarcus GFP or a Pleuromamma, Gaussia or Renilla mulleri
luciferase, that may be present in a biological sample or a
solution derived from such a biological sample.
DESCRIPTION OF THE FIGURES
[0075] FIG. 1 depicts phylogenetic relationships among the
anthozoan GFPs.
[0076] FIGS. 2A-D illustrate the underlying principle of
Bioluminescent Resonance Energy Transfer (BRET) and its use as
sensor: A) in isolation, a luciferase, preferably an anthozoan
luciferase, emits blue light from the coelenterazine-derived
chromophore; B) in isolation, a GFP, preferably an anthozoan GFP
that binds to the luciferase, that is excited with blue-green light
emits green light from its integral peptide based fluorophore; C)
when the luciferase and GFP associate as a complex in vivo or in
vitro, the luciferase non-radiatively transfers its reaction energy
to the GFP flurophore, which then emits the green light; D) any
molecular interaction that disrupts the luciferase-GFP complex can
be quantitatively monitored by observing the spectral shift from
green to blue light.
[0077] FIG. 3 illustrates exemplary BRET sensor architecture.
[0078] FIG. 4 depicts the substitution of altered fluorophores into
the background of Ptilosarcus, Renilla mulleri and Renilla
reniformis GFPs (the underlined regions correspond to amino acids
56-75 of SEQ ID NO. 27 Renilla reniformis GFP; amino acids 59-78 of
SEQ ID NO. 16 Renilla mulleri GFP; and amino acids 9-78 of SEQ ID
NO. 32 for Ptilosarcus GFP).
[0079] FIG. 5 depicts the three anthozoan fluorescent proteins for
which a crystal structure exists; another available commercially
from Clontech as dsRed (from Discosoma striata; also known as
drFP583, as in this alignment); a dark gray background depicts
amino acid conservation, and a light gray background depicts shared
physicochemical properties.
[0080] FIG. 6 compares the sequences of a variety of GFPs,
identifying sites for mutation to reduce multimerization;
abbreviations are as follows: Amemonia majona is amFP486; Zoanthus
sp. zFP506 and zFP538; Discosoma sp. "red" is drFP583; Clavularia
sp. is cFP484; and the GFP from the anthozoal A. sulcata is
designated FP595.
DETAILED DESCRIPTION OF THE INVENTION
[0081] A. Definitions
[0082] B. Fluorescent Proteins
[0083] 1. Green and Blue Fluorescent Proteins
[0084] 2. Renilla reniformis GFP
[0085] C. Bioluminescence Generating Systems and Components
[0086] 1. General Description
[0087] a. Luciferases
[0088] b. Luciferins
[0089] c. Activators
[0090] d. Reactions
[0091] 2. The Renilla System
[0092] 3. Ctenophore Systems
[0093] 4. The aequorin System
[0094] a. Aequorin and Related Photoproteins
[0095] b. Luciferin
[0096] 5. Crustacean, Particularly Cyrpidina Systems
[0097] a. Vargula luciferase
[0098] (1) Purification from Cypridina
[0099] (2) Preparation by Recombinant Methods
[0100] b. Vargula luciferin
[0101] c. Reaction
[0102] 6. Insect Bioluminescent Systems Including Fireflies, Click
Beetles, and Other Insect System
[0103] a. Luciferase
[0104] b. Luciferin
[0105] c. Reaction
[0106] 7. Other Systems
[0107] a. Bacterial Systems
[0108] (1) Luciferases
[0109] (2) Luciferins
[0110] (3) Reactions
[0111] b. Dinoflagellate Bioluminescence Generating Systems
[0112] D. Isolation and Identification of Nucleic Acids Encoding
Luciferases and GFPs
[0113] 1. Isolation of Specimens of the Genus Renilla
[0114] 2. Preparation of Renilla cDNA Expression Libraries
[0115] a. RNA Isolation and cDNA Synthesis
[0116] b. Construction of cDNA Expression Libraries
[0117] 3. Cloning of Renilla reniformis Green Fluorescent
Protein
[0118] 4. Isolation and Identification of DNA Encoding Renilla
mulleri GFP
[0119] 5. Isolation and Identification of DNA Encoding Renilla
mulleri luciferase
[0120] E. Recombinant Expression of Proteins
[0121] 1. DNA encoding Renilla Proteins
[0122] 2. DNA Constructs for Recombinant Production of Renilla
reniformis and Other Proteins
[0123] 3. Host Organisms for Recombinant Production of Renilla
Proteins
[0124] 4. Methods for Recombinant Production of Renilla
Proteins
[0125] 5. Recombinant Cells Expressing Heterologous Nucleic Acid
Encoding luciferases and GFPs
[0126] F. Compositions and Conjugates
[0127] 1. Renilla GFP Compositions
[0128] 2. Renilla luciferase Compositions
[0129] 3. Conjugates
[0130] a. Linkers
[0131] b. Targeting Agents
[0132] c. Anti-tumor Antigen Antibodies
[0133] d. Preparation of the Conjugates
[0134] 4. Formulation of the Compositions for use in the Diagnostic
Systems
[0135] a. The First Composition: Formulation of the Conjugates
[0136] b. The Second Composition
[0137] c. Practice of the Reactions in Combination with Targeting
Agents
[0138] G. Combinations
[0139] H. Exemplary uses of Renilla reniformis GFPs and Encoding
Nucleic Acid Molecules
[0140] 1. Methods for Diagnosis of Neoplasms and Other Tissues
[0141] 2. Methods of Diagnosing Diseases
[0142] 3. Methods for Generating Renilla mulleri luciferase,
Pleuromamma luciferase and Gaussia luciferase Fusion Proteins with
Renilla reniformis GFP
[0143] 4. Cell-Based Assays for Identifying Compounds
[0144] I. Kits
[0145] J. Muteins
[0146] 1. Mutation of GFP Surfaces to Disrupt Multimerization
[0147] 2. Use of Advantageous GFP Surfaces with Substituted
Fluorophores
[0148] K. Transgenic Plants and Animals
[0149] L. Bioluminescence Resonance Energy Transfer (BRET)
System
[0150] 1. Design of Sensors Based on BRET
[0151] 2. BRET Sensor Architectures
[0152] 3. Advantages of BRET Sensors
[0153] A. Definitions
[0154] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents,
applications and publications of referred to throughout the
disclosure are incorporated by reference in their entirety.
[0155] As used herein, chemiluminescence refers to a chemical
reaction in which energy is specifically channeled to a molecule
causing it to become electronically excited and subsequently to
release a photon thereby emitting visible light. Temperature does
not contribute to this channeled energy. Thus, chemiluminescence
involves the direct conversion of chemical energy to light
energy.
[0156] As used herein, luminescence refers to the detectable
electromagnetic (EM) radiation, generally, ultraviolet (UV),
infrared (IR) or visible EM radiation that is produced when the
excited product of an exergic chemical process reverts to its
ground state with the emission of light. Chemiluminescence is
luminescence that results from a chemical reaction. Bioluminescence
is chemiluminescence that results from a chemical reaction using
biological molecules (or synthetic versions or analogs thereof) as
substrates and/or enzymes.
[0157] As used herein, bioluminescence, which is a type of
chemiluminescence, refers to the emission of light by biological
molecules, particularly proteins. The essential condition for
bioluminescence is molecular oxygen, either bound or free in the
presence of an oxygenase, a luciferase, which acts on a substrate,
a luciferin. Bioluminescence is generated by an enzyme or other
protein (luciferase) that is an oxygenase that acts on a substrate
luciferin (a bioluminescence substrate) in the presence of
molecular oxygen and transforms the substrate to an excited state,
which upon return to a lower energy level releases the energy in
the form of light.
[0158] As used herein, the substrates and enzymes for producing
bioluminescence are generically referred to as luciferin and
luciferase, respectively. When reference is made to a particular
species thereof, for clarity, each generic term is used with the
name of the organism from which it derives, for example, bacterial
luciferin or firefly luciferase.
[0159] As used herein, luciferase refers to oxygenases that
catalyze a light emitting reaction. For instance, bacterial
luciferases catalyze the oxidation of flavin mononucleotide (FMN)
and aliphatic aldehydes, which reaction produces light. Another
class of luciferases, found among marine arthropods, catalyzes the
oxidation of Cypridina (Vargula) luciferin, and another class of
luciferases catalyzes the oxidation of Coleoptera luciferin.
[0160] Thus, luciferase refers to an enzyme or photoprotein that
catalyzes a bioluminescent reaction (a reaction that produces
bioluminescence). The luciferases, such as firefly and Gaussia and
Renilla luciferases, that are enzymes which act catalytically and
are unchanged during the bioluminescence generating reaction. The
luciferase photoproteins, such as the aequorin photoprotein to
which luciferin is non-covalently bound, are changed, such as by
release of the luciferin, during bioluminescence generating
reaction. The luciferase is a protein that occurs naturally in an
organism or a variant or mutant thereof, such as a variant produced
by mutagenesis that has one or more properties, such as thermal
stability, that differ from the naturally-occurring protein.
Luciferases and modified mutant or variant forms thereof are well
known. For purposes herein, reference to luciferase refers to
either the photoproteins or luciferases.
[0161] Thus, reference, for example, to "Gaussia luciferase" means
an enzyme isolated from member of the genus Gaussia or an
equivalent molecule obtained from any other source, such as from
another related copepod, or that has been prepared synthetically.
It is intended to encompass Gaussia luciferases with conservative
amino acid substitutions that do not substantially alter activity.
Suitable conservative substitutions of amino acids are known to
those of skill in this art and may be made generally without
altering the biological activity of the resulting molecule. Those
of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The
Bejacmin/Cummings Pub. co., p.224).
[0162] "Renilla GFP" refers to GFPs from the genus Renilla and to
mutants or variants thereof. It is intended to encompass Renilla
GFPs with conservative amino acid substitutions that do not
substantially alter activity and physical properties, such as the
emission spectra and ability to shift the spectral output of
bioluminescence generating systems.
[0163] Such substitutions are preferably made in accordance with
those set forth in TABLE 1 as follows:
1 TABLE 1 Conservative Original Residue Substitution Ala (A) Gly;
Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)
Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile;
Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu;
Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V)
Ile; Leu
[0164] Other substitutions are also permissible and may be
determined empirically or in accord with known conservative
substitutions.
[0165] The luciferases and luciferin and activators thereof are
referred to as bioluminescence generating reagents or components.
Typically, a subset of these reagents will be provided or combined
with an article of manufacture. Bioluminescence will be produced
upon contacting the combination with the remaining reagents. Thus,
as used herein, the component luciferases, luciferins, and other
factors, such as O.sub.2, Mg.sup.2+, Ca.sup.2+ are also referred to
as bioluminescence generating reagents (or agents or
components).
[0166] As used herein, a Renilla reniformis green fluorescent
protein (GFP) refers to a fluorescent protein that is encoded by a
sequence of nucleotides that encodes the protein of SEQ ID NO. 27
or to a green fluorescent protein from Renilla reniformis having at
least 80%, 90% or 95% or greater sequence identity thereto; or that
is encoded by a sequence of nucleotides that hybridizes under high
stringency along its full length to the coding portion of the
sequence of nucleotides set forth in any of SEQ ID NOs. 23-25. A
Renilla reniformis GFP is protein that is fluorescent and is
produced in a Renilla reniformis.
[0167] As used herein, bioluminescence substrate refers to the
compound that is oxidized in the presence of a luciferase, and any
necessary activators, and generates light. These substrates are
referred to as luciferins herein, are substrates that undergo
oxidation in a bioluminescence reaction. These bioluminescence
substrates include any luciferin or analog thereof or any synthetic
compound with which a luciferase interacts to generate light.
Preferred substrates are those that are oxidized in the presence of
a luciferase or protein in a light-generating reaction.
Bioluminescence substrates, thus, include those compounds that
those of skill in the art recognize as luciferins. Luciferins, for
example, include firefly luciferin, Cypridina (also known as
Vargula) luciferin (coelenterazine), bacterial luciferin, as well
as synthetic analogs of these substrates or other compounds that
are oxidized in the presence of a luciferase in a reaction the
produces bioluminescence.
[0168] As used herein, capable of conversion into a bioluminescence
substrate means susceptible to chemical reaction, such as oxidation
or reduction, that yields a bioluminescence substrate. For example,
the luminescence producing reaction of bioluminescent bacteria
involves the reduction of a flavin mononucleotide group (FMN) to
reduced flavin mononucleotide (FMNH.sub.2) by a flavin reductase
enzyme. The reduced flavin mononucleotide (substrate) then reacts
with oxygen (an activator) and bacterial luciferase to form an
intermediate peroxy flavin that undergoes further reaction, in the
presence of a long-chain aldehyde, to generate light. With respect
to this reaction, the reduced flavin and the long chain aldehyde
are substrates.
[0169] As used herein, a bioluminescence generating system refers
to the set of reagents required to conduct a bioluminescent
reaction. Thus, the specific luciferase, luciferin and other
substrates, solvents and other reagents that may be required to
complete a bioluminescent reaction form a bioluminescence system.
Thus a bioluminescence generating system refers to any set of
reagents that, under appropriate reaction conditions, yield
bioluminescence. Appropriate reaction conditions refers to the
conditions necessary for a bioluminescence reaction to occur, such
as pH, salt concentrations and temperature. In general,
bioluminescence systems include a bioluminescence substrate,
luciferin, a luciferase, which includes enzymes luciferases and
photoproteins, and one or more activators. A specific
bioluminescence system may be identified by reference to the
specific organism from which the luciferase derives; for example,
the Vargula (also called Cypridina) bioluminescence system (or
Vargula system) includes a Vargula luciferase, such as a luciferase
isolated from the ostracod, Vargula or produced using recombinant
means or modifications of these luciferases. This system would also
include the particular activators necessary to complete the
bioluminescence reaction, such as oxygen and a substrate with which
the luciferase reacts in the presence of the oxygen to produce
light.
[0170] The luciferases provided herein may be incorporated into
bioluminescence generating systems and used, as appropriate, with
the GFPs provided herein or with other GFPs. Similarly, the GFPs
provided herein may be used with known bioluminescence generating
systems.
[0171] As used herein, the amino acids, which occur in the various
amino acid sequences appearing herein, are identified according to
their well-known, three-letter or one-letter abbreviations. The
nucleotides, which occur in the various DNA molecules, are
designated with the standard single-letter designations used
routinely in the art.
[0172] As used herein, a fluorescent protein refers to a protein
that possesses the ability to fluoresce (i.e., to absorb energy at
one wavelength and emit it at another wavelength). These proteins
can be used as a fluorescent label or marker and in any
applications in which such labels would be used, such as
immunoassays, CRET, FRET, and FET assays, and in the assays
designated herein as BRET assays. For example, a green fluorescent
protein refers to a polypeptide that has a peak in the emission
spectrum at about 510 nm.
[0173] As used herein, the term BRET (Bioluminescence Resonance
Energy Transfer) refers to non-radiative luciferase-to-FP energy
transfer. It differs from (Fluorescence Resonance Energy Transfer),
which refers to energy transfer between chemical fluors.
[0174] As used herein, a BRET system refers the combination of a
FP, in this case Renilla reniformis GFP and a luciferase for
resonance energy transfer. BRET refers to any method in which the
luciferase is used to generate the light upon reaction with a
luciferin which is then non-radiatively transferred to a FP. The
energy is transferred to a FP, particularly a GFP, which focuses
and shifts the energy and emits it at a different wavelength. In
preferred embodiments, the BRET system includes a bioluminescence
generating system and a Renilla reniformis GFP. The bioluminescence
generating system is preferably a Renilla system. Hence, the
preferred pair is a Renilla luciferase and a Renilla GFP, which
specifically interact. Alterations in the binding will be reflected
in changes in the emission spectra of light produced by the
luciferase. As a result the pair can function as a sensor of
external events.
[0175] As used herein, a biosensor (or sensor) refers to a BRET
system for use to detect alterations in the environment in vitro or
in vivo in which the BRET system is used.
[0176] As used herein, modulator with reference to a BRET system
refers to a molecule or molecules that undergo a conformation
change in response to interaction with another molecule thereby
affecting the proximity and/or orientation of the GFP and
luciferase in the BRET system. Modulators include, but are not
limited to, a protease site, a second messenger binding site, an
ion binding molecule, a receptor, an oligomer, an enzyme substrate,
a ligand, or other such binding molecule. If the GFP and luciferase
are each linked to the modulator, changes in conformation alter the
spacial relationship between the GFP and luciferase. The modulator
can be a single entity covalently attached to one or both of the
luciferase and GFP; it can be two separate entities each linked to
either the luciferase or GFP. The modulator(s), GFP and luciferase
can be a single fusion protein, or a fusion protein of at least two
of the entities. The components can be chemically linked, such as
through thiol or disulfide linkages, using linkers as provided
herein. The GFP and luciferase can be linked directly or via
linker, which can be a chemical linkage.
[0177] As used herein, "not strictly catalytically" means that the
photoprotein acts as a catalyst to promote the oxidation of the
substrate, but it is changed in the reaction, since the bound
substrate is oxidized and bound molecular oxygen is used in the
reaction. Such photoproteins are regenerated by addition of the
substrate and molecular oxygen under appropriate conditions known
to those of skill in this art.
[0178] As used herein, "nucleic acid" refers to a polynucleotide
containing at least two covalently linked nucleotide or nucleotide
analog subunits. A nucleic acid can be a deoxyribonucleic acid
(DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA.
Nucleotide analogs are commercially available and methods of
preparing polynucleotides containing such nucleotide analogs are
known (Lin et al. (1994) Nucl. Acids Res. 22:5220-5234; Jellinek et
al. (1995) Biochemistry 34:11363-11372; Pagratis et al. (1997)
Nature Biotechnol. 15:68-73). The nucleic acid can be
single-stranded, double-stranded, or a mixture thereof For purposes
herein, unless specified otherwise, the nucleic acid is
double-stranded, or it is apparent from the context.
[0179] As used herein, a second messenger includes, but are not
limited to, cAMP, cGMP, inositol phosphates, such as IP2 and IP3,
NO (nitric oxide), Ca.sup.2+, ceramide; DAG and arachidonic
acid.
[0180] Hence, the term "nucleic acid" refers to single-stranded
and/or double-stranded polynucleotides, such as deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA), as well as analogs or
derivatives of either RNA or DNA. Also included in the term
"nucleic acid" are analogs of nucleic acids such as peptide nucleic
acid (PNA), phosphorothioate DNA, and other such analogs and
derivatives.
[0181] As used herein, the term "nucleic acid molecule" and
"nucleic acid fragment" are used interchangeably.
[0182] As used herein, DNA is meant to include all types and sizes
of DNA molecules including cDNA, plasmids and DNA including
modified nucleotides and nucleotide analogs.
[0183] As used herein, nucleotides include nucleoside mono-, di-,
and triphosphates. Nucleotides also include modified nucleotides,
such as, but are not limited to, phosphorothioate nucleotides and
deazapurine nucleotides and other nucleotide analogs.
[0184] As used herein, a nucleic acid probe is single-stranded DNA
or RNA that has a sequence of nucleotides that includes at least 14
contiguous bases, preferably at least 16 contiguous bases,
typically about 30, that are the same as (or the complement of) any
14 or more contiguous bases set forth in any of SEQ ID NOs. 23-25
and herein. Among the preferred regions from which to construct
probes include 5' and/or 3' coding sequences, sequences predicted
to encode regions that are conserved among Renilla species. Probes
from regions conserved among Renilla species GFPs are for isolating
GFP-encoding nucleic acid from Renilla libraries.
[0185] In preferred embodiments, the nucleic acid probes are
degenerate probes of at least 14 nucleotides, preferably 16 to 30
nucleotides, are provided.
[0186] In preferred embodiments, the nucleic acid probes are
degenerate probes of at least 14 nucleotides, preferably 16 to 30
nucleotides, that are based on amino acids of Renilla reniformis
set forth in above.
[0187] As used herein, vector (or plasmid) refers to discrete
elements that are used to introduce heterologous DNA into cells for
either expression or replication thereof. Selection and use of such
vehicles are well within the skill of the artisan. An expression
vector includes vectors capable of expressing DNA operatively
linked with regulatory sequences, such as promoter regions, that
are capable of effecting expression of such DNA molecules. Thus, an
expression vector refers to a recombinant DNA or RNA construct,
such as a plasmid, a phage, recombinant virus or other vector that,
upon introduction into an appropriate host cell, results in
expression of the cloned DNA. Appropriate expression vectors are
well known to those of skill in the art and include those that are
replicable in eukaryotic cells and/or prokaryotic cells and those
that remain episomal or those which integrate into the host cell
genome. Presently preferred plasmids for expression of Gaussia
luciferase, Renilla GFP and luciferase are those that are expressed
in bacteria and yeast, such as those described herein.
[0188] As used herein, a promoter region or promoter element refers
to a segment of DNA or RNA that controls transcription of the DNA
or RNA to which it is operatively linked. The promoter region
includes specific sequences that are sufficient for RNA polymerase
recognition, binding and transcription initiation. This portion of
the promoter region is referred to as the promoter. In addition,
the promoter region includes sequences that modulate this
recognition, binding and transcription initiation activity of RNA
polymerase. These sequences may be cis acting or may be responsive
to trans acting factors. Promoters, depending upon the nature of
the regulation, may be constitutive or regulated. Exemplary
promoters contemplated for use in prokaryotes include the
bacteriophage T7 and T3 promoters, and the like.
[0189] As used herein, operatively linked or operationally
associated refers to the functional relationship of DNA with
regulatory and effector sequences of nucleotides, such as
promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences. For example, operative linkage of DNA
to a promoter refers to the physical and functional relationship
between the DNA and the promoter such that the transcription of
such DNA is initiated from the promoter by an RNA polymerase that
specifically recognizes, binds to and transcribes the DNA. In order
to optimize expression and/or in vitro transcription, it may be
necessary to remove, add or alter 5' untranslated portions of the
clones to eliminate extra, potentially inappropriate alternative
translation initiation (i.e., start) codons or other sequences that
may interfere with or reduce expression, either at the level of
transcription or translation. Alternatively, consensus ribosome
binding sites (see, e.g., Kozak (I991) J. Biol. Chem.
266:19867-19870) can be inserted immediately 5' of the start codon
and may enhance expression. The desirability of (or need for) such
modification may be empirically determined.
[0190] As used herein, to target a targeted agent, such as a
luciferase, means to direct it to a cell that expresses a selected
receptor or other cell surface protein by linking the agent to a
such agent. Upon binding to or interaction with the receptor or
cell surface protein, the targeted agent can be reacted with an
appropriate substrate and activating agents, whereby bioluminescent
light is produced and the tumorous tissue or cells distinguished
from non-tumorous tissue.
[0191] As used herein, an effective amount of a compound for
treating a particular disease is an amount that is sufficient to
ameliorate, or in some manner reduce the symptoms associated with
the disease. Such amount may be administered as a single dosage or
may be administered according to a regimen, whereby it is
effective. The amount may cure the disease but, typically, is
administered in order to ameliorate the symptoms of the disease.
Repeated administration may be required to achieve the desired
amelioration of symptoms.
[0192] As used herein, an effective amount of a conjugate for
diagnosing a disease is an amount that will result in a detectable
tissue. The tissues are detected by visualization either without
aid from a detector more sensitive than the human eye, or with the
use of a light source to excite any fluorescent products.
[0193] As used herein, visualizable means detectable by eye,
particularly during surgery under normal surgical conditions, or,
if necessary, slightly dimmed light.
[0194] As used herein, pharmaceutically acceptable salts, esters or
other derivatives of the conjugates include any salts, esters or
derivatives that may be readily prepared by those of skill in this
art using known methods for such derivatization and that produce
compounds that may be administered to animals or humans without
substantial toxic effects and that either are pharmaceutically
active or are prodrugs.
[0195] As used herein, treatment means any manner in which the
symptoms of a conditions, disorder or disease are ameliorated or
otherwise beneficially altered. Treatment also encompasses any
pharmaceutical use of the compositions herein.
[0196] As used herein, amelioration of the symptoms of a particular
disorder by administration of a particular pharmaceutical
composition refers to any lessening, whether permanent or
temporary, lasting or transient that can be attributed to or
associated with administration of the composition.
[0197] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound may, however,
be a mixture of stereoisomers or isomers. In such instances,
further purification might increase the specific activity of the
compound.
[0198] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound will be
regenerated by metabolic processes. The prodrug may be designed to
alter the metabolic stability or the transport characteristics of a
drug, to mask side effects or toxicity, to improve the flavor of a
drug or to alter other characteristics or properties of a drug. By
virtue of knowledge of pharmacodynamic processes and drug
metabolism in vivo, those of skill in this art, once a
pharmaceutically active compound is known, can design prodrugs of
the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A
Biochemical Approach, Oxford University Press, New York, pages
388-392).
[0199] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures. Biological activities may be observed in in vitro systems
designed to test or use such activities. Thus, for purposes herein
the biological activity of a luciferase is its oxygenase activity
whereby, upon oxidation of a substrate, light is produced.
[0200] As used herein, targeting agent (TA) refers to an agent that
specifically or preferentially targets a linked targeted agent, a
luciferin or luciferase, to a neoplastic cell or tissue.
[0201] As used herein, tumor antigen refers to a cell surface
protein expressed or located on the surface of tumor cells.
[0202] As used herein, neoplastic cells include any type of
transformed or altered cell that exhibits characteristics typical
of transformed cells, such as a lack of contact inhibition and the
acquisition of tumor-specific antigens. Such cells include, but are
not limited to leukemic cells and cells derived from a tumor.
[0203] As used herein, neoplastic disease is any disease in which
neoplastic cells are present in the individual afflicted with the
disease. Such diseases include, any disease characterized as
cancer.
[0204] As used herein, metastatic tumors refers to tumors that are
not localized in one site.
[0205] As used herein, specialty tissue refers to non-tumorous
tissue for which information regarding location is desired. Such
tissues include, for example, endometriotic tissue, ectopic
pregnancies, tissues associated with certain disorders and
myopathies or pathologies.
[0206] As used herein, a receptor refers to a molecule that has an
affinity for a given ligand. Receptors may be naturally-occurring
or synthetic molecules. Receptors may also be referred to in the
art as anti-ligands. As used herein, the receptor and anti-ligand
are interchangeable. Receptors can be used in their unaltered state
or as aggregates with other species. Receptors may be attached,
covalently or noncovalently, or in physical contact with, to a
binding member, either directly or indirectly via a specific
binding substance or linker. Examples of receptors, include, but
are not limited to: antibodies, cell membrane receptors surface
receptors and internalizing receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells, or other materials), drugs, polynucleotides,
nucleic acids, peptides, cofactors, lectins, sugars,
polysaccharides, cells, cellular membranes, and organelles.
[0207] Examples of receptors and applications using such receptors,
include but are not restricted to:
[0208] a) enzymes: specific transport proteins or enzymes essential
to survival of microorganisms, which could serve as targets for
antibiotic (ligand) selection;
[0209] b) antibodies: identification of a ligand-binding site on
the antibody molecule that combines with the epitope of an antigen
of interest may be investigated; determination of a sequence that
mimics an antigenic epitope may lead to the development of vaccines
of which the immunogen is based on one or more of such sequences or
lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune
diseases
[0210] c) nucleic acids: identification of ligand, such as protein
or RNA, binding sites;
[0211] d) catalytic polypeptides: polymers, preferably
polypeptides, that are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products; such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, in which the
functionality is capable of chemically modifying the bound reactant
(see, e.g., U.S. Pat. No. 5,215,899);
[0212] e) hormone receptors: determination of the ligands that bind
with high affinity to a receptor is useful in the development of
hormone replacement therapies; for example, identification of
ligands that bind to such receptors may lead to the development of
drugs to control blood pressure; and
[0213] f) opiate receptors: determination of ligands that bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0214] As used herein, antibody includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain.
[0215] As used herein, an antibody conjugate refers to a conjugate
in which the targeting agent is an antibody.
[0216] As used herein, antibody activation refers to the process
whereby activated antibodies are produced. Antibodies are activated
upon reaction with a linker, such as heterobifunctional
reagent.
[0217] As used herein, a surgical viewing refers to any procedure
in which an opening is made in the body of an animal. Such
procedures include traditional surgeries and diagnostic procedures,
such as laparoscopies and arthroscopic procedures.
[0218] As used herein, humanized antibodies refer to antibodies
that are modified to include "human" sequences of amino acids so
that administration to a human will not provoke an immune response.
Methods for preparation of such antibodies are known. For example,
the hybridoma that expresses the monoclonal antibody is altered by
recombinant DNA techniques to express an antibody in which the
amino acid composition of the non-variable regions is based on
human antibodies. Computer programs have been designed to identify
such regions.
[0219] As used herein, ATP, AMP, NAD+ and NADH refer to adenosine
triphosphate, adenosine monophosphate, nicotinamide adenine
dinucleotide (oxidized form) and nicotinamide adenine dinucleotide
(reduced form), respectively.
[0220] As used herein, production by recombinant means by using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned
DNA.
[0221] As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
[0222] As used herein equivalent, when referring to two sequences
of nucleic acids means that the two sequences in question encode
the same sequence of amino acids or equivalent proteins. When
"equivalent" is used in referring to two proteins or peptides, it
means that the two proteins or peptides have substantially the same
amino acid sequence with only conservative amino acid substitutions
(see, e.g., Table 1, above) that do not substantially alter the
activity or function of the protein or peptide. When "equivalent"
refers to a property, the property does not need to be present to
the same extent (e.g., two peptides can exhibit different rates of
the same type of enzymatic activity), but the activities are
preferably substantially the same. "Complementary," when referring
to two nucleotide sequences, means that the two sequences of
nucleotides are capable of hybridizing, preferably with less than
25%, more preferably with less than 15%, even more preferably with
less than 5%, most preferably with no mismatches between opposed
nucleotides. Preferably the two molecules will hybridize under
conditions of high stringency.
[0223] As used herein: stringency of hybridization in determining
percentage mismatch is as follows:
[0224] 1) high stringency: 0.1.times.SSPE, 0.1% SDS, 65.degree.
C.
[0225] 2) medium stringency: 0.2.times.SSPE, 0.1% SDS, 50.degree.
C.
[0226] 3) low stringency: 1.0.times.SSPE, 0.1% SDS, 50.degree.
C.
[0227] It is understood that equivalent stringencies may be
achieved using alternative buffers, salts and temperatures.
[0228] The term "substantially" identical or homologous or similar
varies with the context as understood by those skilled in the
relevant art and generally means at least 70%, preferably means at
least 80%, more preferably at least 90%, and most preferably at
least 95% identity. The terms "homology" and "identity" are often
used interchangeably. In general, sequences are aligned so that the
highest order match is obtained (see, e.g.: Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991;
Carillo et al. (1988) SIAM J Applied Math 48:1073).
[0229] By sequence identity, the number of conserved amino acids
are determined by standard alignment algorithms programs, and are
used with default gap penalties established by each supplier.
Substantially homologous nucleic acid molecules would hybridize
typically at moderate stringency or at high stringency all along
the length of the nucleic acid of interest. Also contemplated are
nucleic acid molecules that contain degenerate codons in place of
codons in the hybridizing nucleic acid molecule.
[0230] Whether any two nucleic acid molecules have nucleotide
sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% "identical" can be determined using known computer algorithms
such as the "FAST A" program, using for example, the default
parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA
85:2444 (other programs include the GCG program package (Devereux,
J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP,
BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990);
Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San
Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math
48:1073). For example, the BLAST function of the National Center
for Biotechnology Information database may be used to determine
identity. Other commercially or publicly available programs
include, DNAStar "MegAlign" program (Madison, Wis.) and the
University of Wisconsin Genetics Computer Group (UWG) "Gap" program
(Madison Wis.)). Percent homology or identity of proteins and/or
nucleic acid moleucles may be determined, for example, by comparing
sequence information using a GAP computer program (e.g., Needleman
et al. (1970) J. Mol Biol. 48:443, as revised by Smith and Waterman
((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines
similarity as the number of aligned symbols (i.e., nucleotides or
amino acids) which are similar, divided by the total number of
symbols in the shorter of the two sequences. Default parameters for
the GAP program may include: (1) a unary comparison matrix
(containing a value of 1 for identities and 0 for non-identities)
and the weighted comparison matrix of Gribskov et al (1986) Nucl.
Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds.,
ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical
Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps.
[0231] Therefore, as used herein, the term "identity" represents a
comparison between a test and a reference polypeptide or
polynucleotide. For example, a test polypeptide may be defined as
any polypeptide that is 90% or more identical to a reference
polypeptide. As used herein, the term at least "90% identical to"
refers to percent identities from 90 to 99.99 relative to the
reference polypeptides. Identity at a level of 90% or more is
indicative of the fact that, assuming for exemplification purposes
a test and reference polynucleotide length of 100 amino acids are
compared. No more than 10% (i.e., 10 out of 100) amino acids in the
test polypeptide differs from that of the reference polypeptides.
Similar comparisons may be made between a test and reference
polynucleotides. Such differences may be represented as point
mutations randomly distributed over the entire length of an amino
acid sequence or they may be clustered in one or more locations of
varying length up to the maximum allowable, e.g. (10/100) amino
acid difference (approximately 90% identity). Differences are
defined as nucleic acid or amino acid substitutions, or deletions.
At level of homologies or identities above about 85-90%, the result
should be independent of the program and gap parameters set; such
high levels of identity readily can be assess, often without
relying on software.
[0232] As used herein, primer refers to an oligonucleotide
containing two or more deoxyribonucleotides or ribonucleotides,
preferably more than three, from which synthesis of a primer
extension product can be initiated. Experimental conditions
conducive to synthesis include the presence of nucleoside
triphosphates and an agent for polymerization and extension, such
as DNA polymerase, and a suitable buffer, temperature and pH.
[0233] As used herein, a composition refers to any mixture. It may
be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0234] As used herein, a combination refers to any association
between two or among more items.
[0235] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0236] Examples of receptors and applications using such receptors,
include but are not restricted to:
[0237] a) enzymes: specific transport proteins or enzymes essential
to survival of microorganisms, which could serve as targets for
antibiotic (ligand) selection;
[0238] b) antibodies: identification of a ligand-binding site on
the antibody molecule that combines with the epitope of an antigen
of interest may be investigated; determination of a sequence that
mimics an antigenic epitope may lead to the development of vaccines
of which the immunogen is based on one or more of such sequences or
lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune
diseases
[0239] c) nucleic acids: identification of ligand, such as protein
or RNA, binding sites;
[0240] d) catalytic polypeptides: polymers, preferably
polypeptides, that are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products; such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, in which the
functionality is capable of chemically modifying the bound reactant
(see, e.g., U.S. Pat. No. 5,215,899);
[0241] e) hormone receptors: determination of the ligands that bind
with high affinity to a receptor is useful in the development of
hormone replacement therapies; for example, identification of
ligands that bind to such receptors may lead to the development of
drugs to control blood pressure; and
[0242] f) opiate receptors: determination of ligands that bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0243] As used herein, complementary refers to the topological
compatibility or matching together of interacting surfaces of a
ligand molecule and its receptor. Thus, the receptor and its ligand
can be described as complementary, and furthermore, the contact
surface characteristics are complementary to each other.
[0244] As used herein, a ligand-receptor pair or complex formed
when two macromolecules have combined through molecular recognition
to form a complex.
[0245] As used herein, a substrate refers to any matrix that is
used either directly or following suitable derivatization, as a
solid support for chemical synthesis, assays and other such
processes. Preferred substrates herein, are silicon substrates or
siliconized substrates that are derivitized on the surface intended
for linkage of anti-ligands and ligands and other macromolecules,
including the fluorescent proteins, phycobiliproteins and other
emission shifters.
[0246] As used herein, a matrix refers to any solid or semisolid or
insoluble support on which the molecule of interest, typically a
biological molecule, macromolecule, organic molecule or biospecific
ligand is linked or contacted. Typically a matrix is a substrate
material having a rigid or semi-rigid surface. In many embodiments,
at least one surface of the substrate will be substantially flat,
although in some embodiments it may be desirable to physically
separate synthesis regions for different polymers with, for
example, wells, raised regions, etched trenches, or other such
topology. Matrix materials include any materials that are used as
affinity matrices or supports for chemical and biological molecule
syntheses and analyses, such as, but are not limited to:
polystyrene, polycarbonate, polypropylene, nylon, glass, dextran,
chitin, sand, pumice, polytetrafluoroethylene, agarose,
polysaccharides, dendrimers, buckyballs, polyacrylamide,
Kieselguhr-polyacrylamide non-covalent composite,
polystyrene-polyacrylamide covalent composite, polystyrene-PEG
(polyethyleneglycol) composite, silicon, rubber, and other
materials used as supports for solid phase syntheses, affinity
separations and purifications, hybridization reactions,
immunoassays and other such applications.
[0247] As used herein, the attachment layer refers the surface of
the chip device to which molecules are linked. Typically, the chip
is a semiconductor device, which is coated on a least a portion of
the surface to render it suitable for linking molecules and inert
to any reactions to which the device is exposed. Molecules are
linked either directly or indirectly to the surface, linkage may be
effected by absorption or adsorption, through covalent bonds, ionic
interactions or any other interaction. Where necessary the
attachment layer is adapted, such as by derivatization for linking
the molecules.
[0248] B. Fluorescent Proteins
[0249] The GFP from Aequorea and that of the sea pansy Renilla
reniformis share the same chromophore, yet Aequorea GFP has two
absorbance peaks at 395 and 475 nm, whereas Renilla GFP has only a
single absorbance peak at 498 nm, with about 5.5 fold greater
monomer extinction coefficient the major 395 mn peak of the
Aequorea protein (Ward, W. W. in Bioluminescence and
Chemiluminescence (eds. DeLuca, M. A. & McElroy, W. D.) 235-242
(Academic Press, New York, 1981)). The spectra of the isolated
chromophore and denatured protein at neutral pH do not match the
spectra of either native protein (Cody, C. W. et al. (1993)
Biochemistry 32:1212-1218).
[0250] 1. Green and Blue Fluorescent Proteins
[0251] As described herein, blue light is produced using the
Renilla luciferase or the Aequorea photoprotein in the presence of
Ca.sup.2+ and the coelenterazine luciferin or analog thereof. This
light can be converted into a green light if a green fluorescent
protein (GFP) is added to the reaction. Green fluorescent proteins,
which have been purified (see, e.g., Prasher et al. (1992) Gene
111:229-233) and also cloned (see, e.g., International PCT
Application No. WO 95/07463, which is based on U.S. application
Ser. No. 08/119,678 and U.S. application Ser. No. 08/192,274, which
are herein incorporated by reference), are used by cnidarians as
energy-transfer acceptors. GFPs fluoresce in vivo upon receiving
energy from a luciferase-oxyluciferein excited-state complex or a
Ca.sup.2+-activated photoprotein. The chromophore is modified amino
acid residues within the polypeptide. The best characterized GFPs
are those of Aequorea and Renilla (see, e.g., Prasher et al. (1992)
Gene 111:229-233; Hart, et al. (1979) Biochemistry 18:2204-2210).
For example, a green fluorescent protein (GFP) from Aequorea
victoria contains 238 amino acids, absorbs blue light and emits
green light. Thus, inclusion of this protein in a composition
containing the aequorin photoprotein charged with coelenterazine
and oxygen, can, in the presence of calcium, result in the
production of green light. Thus, it is contemplated that GFPs may
be included in the bioluminescence generating reactions that employ
the aequorin or Renilla luciferases or other suitable luciferase in
order to enhance or alter color of the resulting
bioluminescence.
[0252] 2. Renilla reniformis GFP
[0253] Purified Renilla reniformis GFP and muteins thereof are
provided. Presently preferred Renilla GFP for use in the
compositions herein is Renilla reniformis GFP having the sequence
of amino acids set forth in SEQ ID NO. 27. The Renilla GFP and GFP
peptides can be isolated from natural sources or isolated from a
prokaryotic or eukaryotic cell transfected with nucleic acid that
encodes the Renilla GFP and/or GFP peptides, such as those encoded
by the sequences of nucleotides set forth in SEQ ID NOs. 23-25.
[0254] The encoding nucleic acid molecules are provided. Preferred
are those that encode the protein having the sequence of amino
acids (SEQ ID NO. 27):
[0255]
mdlaklglkevmptkinleglvgdhafsmegvgegnilegtqevkisvtkgapipfafdivsv
afsygnraytgypeeisdyflqsfpegftyerniryqdggtaivksdisledgkfivnvdfkakdl
rrmgpvmqqdivgmqpsyesmytnvtsvigeciiafklqtgkhftyhmrtvykskkpvet
mplyhfiqhrlvktnvdtasgyvvqhetaiaahstikkiegsip,
[0256] and is preferably the sequence set forth in SEQ ID NO.
26.
[0257] In particular, nucleic acid molecules encoding a Renilla
reniformis GFP having any of the following sequences are provided
(see SEQ ID NOs. 23-25):
2 2 Renilla renformis GFP Clone-1 GGCACGAGGGTTTCCTGACACAATA- AA-
AACCTTTCAAATTGTTTCTC TGTAGCAGTAAGTATGGATCTCGCAAAACTTGG- TTTG-
AAGGAAGTG ATGCCTACTAAAATCAACTTAGAAGGACTGGTTGGCGACCAC- GCTT
TCTCAATGGAAGGAGTTGGCGAAGGCAACATATTGGAAGGAACTCA
AGAGGTGAAGATATCGGTAACAAAAGGCGCACCACTCCCATTCGC
ATTTGATATCGTATCTGTGGCTTTTTCATATGGGAACAGAGCTTA
TACCGGTTACCCAGAAGAAATTTCCGACTACTTCCTCCAGTCGTT
TCCAGAAGGCTTTACTTACGAGAGAAACATTCGTTATCA
AGATGGAGGAACTGCAATTGTTAAATCTGATATAAGCTTGGAA
GATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAAGGATCT
ACGTCGCATGGGACCAGTCATGCAGCAAGACATCGTGGGTATGCA
GCCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGGGA
ATGTATAATAGCATTCAAACTTCAAACTGGCAAGCATTTCACTTACCA
CATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCA
TTGTATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACA
CAGCCAGTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTC
TACAATCAAAAAAATTGAAGGCTCTTTACCATAGATACCTGTACACAAT
TATTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAAT
AAAATATTAAATATAAAAAAAAAAAAAAAAAAAAAAAA; Renilla renformis GFP
Clone-2 GGCACGAGGCTGACACAATAAAAAACCTTTCAAATTGTTTCTCTGTAGC
AGGAAGTATGGATCTCGCAAAACTTGGTTTGAAGGAAGTGATGCCTACT
AAAATCAACTTAGAAGGACTGGTTGGCGACCACGCTTTCTCAATGGAAG
GAGTTGGCGAAGGCAACATATTGGAAGGAACTCAAGAGGTGAAGATAT
CGGTAACAAAAGGCGCACCACTCCCATTCGCATTTGATATCGTATCTGT
TGCTTTCTCATATGGGAACAGAGCTTATACTGGTTACCCAGAAGAAATT
TCCGACTACTTCCTCCAGTCGTTTCCAGAAGGCTTTACTTACGAGAGAA
ACATTCGTTATCAAGATGGAGGAACTGCAATTGTTAAATCTGATATAAG
CTTGGAAGATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAAGGAT
CTACGTCGCATGGGACCAGTCATGCAGCAAGACATCGTGGGTATGCAG
CCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGGGA
ATGTATAATAGCATTCAAACTTCAAACTGGCAAACATTTCACTTACCAC
ATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCATTG
TATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACACAGCCA
GTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTCTACAAT
CAAAAAAATTGAAGGCTCTTTACCATAGATATCTATACACAATTA
TTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAATAA
AATATTAAATATAAAAAAAAAAAAAAAAAAAAAAA; and Renilla renformis GEP
Clone-3 GGCACGAGGGTTTCCTGACACAATAAAAACCTTTCAAATTGTTT- -CTCTG
TAGCAGTAAGTATGGATCTCGCAAAACTTGGTTTGAAGGAAGTGATGCC
TACTAAAATCAACTTAGAAGGACTGGTTGGCGACCACGCTTTCTCAATG
GAAGGAGTTGGCGAAGGCAACATATTGGAAGGAACTCAAGAGGTGAAG
ATATCGGTAACAAAAGGCGCACCACTCCCATTCGCATTTGATATCGTAT
CTGTGGCTTTTTCATATGGGAACAGAGCTTATACCGGTTACCCAGAAGA
AATTTCCGACTACTTCCTCCAGTCGTTTCCAGAAGGCTTTACTTACGAGA
GAAACATTCGTTATCAAGATGGAGGAACTGCAATTGTTAAATCTGATAT
AAGCTTGGAAGATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAA
GGATCTACGTCGCATGGGACCAGTGATGCAGCAAGACATCGTGGGTAT
GCAGCCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGG
GAATGTATAATAGCATTCAAACTTCAAACTGGCAAGCATTTCACTTACC
ACATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCAT
TGTATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACACAGC
CAGTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTCTACA
ATCAAAAAAATTGAAGGCTCTTTACCATAGATACCTGTACACAATTA
TTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAATAA
AATATTAAATATATGCTTTTGCAAAAAAAAAAAAAAAAAAAAAA
[0258] are provided.
[0259] An exemplary mutein is set forth in SEQ ID NO. 33, and
humanized codon are set forth in SEQ ID NO. 26.
[0260] Also contemplated are the coding portion of the sequence of
nucleotides that hybridize under moderate or high stringency to the
sequence of nucleotides set forth above, particularly when using
probes provided herein. Probes derived from this nucleic acid that
can be used in methods provided herein to isolate GFPs from any
Renilla reniformis species are provided. In an exemplary
embodiment, nucleic acid encoding Renilla reniformis GFP is
provided. This nucleic acid encodes the sequence of amino acids set
forth above.
[0261] GFPs, including the Renilla reniformis protein provided
herein, are activated by blue light to emit green light and thus
may be used in the absence of luciferase and in conjunction with an
external light source with novelty items (see U.S. Pat. Nos.
5,876,995, 6,152,358 and 6,113,886) and in conjunction with
bioluminescence generating system for novelty items (see U.S. Pat.
Nos. 5,876,995, 6,152,358 and 6,113,886), for tumor diagnosis (see,
allowed co-pending U.S. application Ser. No. 08/908,909) and in
biochips (see, U.S. application Ser. No. 08/990,103, which is
published as International PCT application No. WO 98/26277).
[0262] Renilla reniformis GFP is intended for use in any of the
novelty items and combinations, such as the foods, including
beverages, greeting cards, and toys, including bubble making toys,
particularly bubble-making compositions or mixtures. Also of
particular interest are the use of these proteins in cosmetics,
particularly face paints or make-up, hair colorants or hair
conditioners, mousses or other such products and skin creams. Such
systems are particularly of interest because no luciferase is
needed to activate the photoprotein and because the proteins are
non-toxic and safe to apply to the skin, hair, eyes and to ingest.
These fluorescent proteins may also be used in addition to
bioluminescence generating systems to enhance or create an array of
different colors. Transgenic animals and plants that express the
Renilla reniformis GFP-encoding nucleic acid are also provided.
Such animals and plants, include transgenic fish, transgenic worms
for use, for example, as lures for fishing; transgenic animals,
such as monkeys and rodents for research in which a marker gene is
used, and transgenic animals as novelty items and to produce
glowing foods, such as ham, eggs, chicken, and other meats;
transgenic plants in which the Renilla reniformis is a marker, and
also transgenic plants that are novelty items, particuarly
ornamental plants, such as glowing orchids, roses and other
flowering plants.
[0263] The Renilla reniformis GFP may be used alone or in
combination with bioluminescence generating systems to produce an
array of colors. They may be used in combinations such that the
color of, for example, a beverage changes over time, or includes
layers of different colors. The cloning and expression of Renilla
reniformis GFP and uses thereof are described below.
[0264] C. Bioluminescence Generating Systems and Components
[0265] The following is a description of bioluminescence generating
systems and the components thereof. The Renilla reniformis GFP
provided herein can be used alone for a variety of applications,
and with any compatible bioluminescence generating systems.
[0266] A bioluminescence-generating system refers to the components
that are necessary and sufficient to generate bioluminescence.
These include a luciferase, luciferin and any necessary co-factors
or conditions. Virtually any bioluminescent system known to those
of skill in the art will be amenable to use in the apparatus,
systems, combinations and methods provided herein. Factors for
consideration in selecting a bioluminescent-generating system,
include, but are not limited to: the targeting agent used in
combination with the bioluminescence; the medium in which the
reaction is run; stability of the components, such as temperature
or pH sensitivity; shelf life of the components; sustainability of
the light emission, whether constant or intermittent; availability
of components; desired light intensity; color of the light; and
other such factors. Such bioluminescence generating systems are
known (see those described in U.S. Pat. Nos. 5,876,995, 6,152,358
and 6,113,886).
[0267] 1. General Description
[0268] In general, bioluminescence refers to an energy-yielding
chemical reaction in which a specific chemical substrate, a
luciferin, undergoes oxidation, catalyzed by an enzyme, a
luciferase. Bioluminescent reactions are easily maintained,
requiring only replenishment of exhausted luciferin or other
substrate or cofactor or other protein, in order to continue or
revive the reaction. Bioluminescence generating reactions are
well-known to those of skill in this art and any such reaction may
be adapted for use in combination with articles of manufacture as
described herein.
[0269] There are numerous organisms and sources of bioluminescence
generating systems, and some representative genera and species that
exhibit bioluminescence are set forth in the following table
(reproduced in part from Hastings in (1995) Cell Physiology: Source
Book, N. Sperelakis (ed.), Academic Press, pp 665-681):
3TABLE 2 Representative Luminous Organism Type of Organism
Representative Genera Bacteria Photobacterium Vibrio Xenorhabdus
Mushrooms Panus, Armillaria Pleurotus Dinoflagellates Gonyaulax
Pyrocystis Noctiluca Cnidaria (coelenterates) Jellyfish Acquorea
Hydroid Obelia Sea Pansy Renilla Ctenophores Mnemiopsis Beroe
Annelids Earthworms Diplocardia Marine polychaetes Chaetopterus,
Phyxotrix Syllid fireworm Odontosyllis Molluscs Limpet Latia Clam
Pholas Squid Heteroteuthis Heterocarpus Crustacea Ostracod Vargula
(Cypridina) Shrimp (euphausids) Meganyctiphanes Acanthophyra
Oplophorus Gnathophausia Decapod Sergestes Copepods Insects
Coleopterids (beetles) Firefly Photinus, Photiuris Click beetles
Pyrophorus Railroad worm Phengodes, Phrixothrix Diptera (flies)
Arachnocampa Echinoderms Brittle stars Ophiopsila Sea cucumbers
Laetmogone Anthozoans Renilla Chordates Tunicates Pyrosoma Fish
Cartilaginos Squalus Bony Ponyfish Leiognathus Flashlight fish
Photoblepharon Angler fish Cryptopsaras Midshipman Porichthys
Latern fish Benia Shiny loosejaw Aristostomias Hatchet fish
Agyropelecus And other fish Pachystomias Malacosteus Midwater fish
Cyclothone Neoscopelus Tarletonbeania
[0270] Other bioluminescent organisms contemplated for use herein
are Gonadostomias, Gaussia (copepods), Watensia, Halisturia,
Vampire squid, Glyphus, Mycotophids (fish), Vinciguerria, Howella,
Florenciella, Chaudiodus, Melanocostus and Sea Pens.
[0271] It is understood that a bioluminescence generating system
may be isolated from natural sources, such as those in the above
Table, or may be produced synthetically. In addition, for uses
herein, the components need only be sufficiently pure so that
mixture thereof, under appropriate reaction conditions, produces a
glow so that cells and tissues can be visualized during a surgical
procedure.
[0272] Thus, in some embodiments, a crude extract or merely
grinding up the organism may be adequate. Generally, however,
substantially pure components are used. Also, components may be
synthetic components that are not isolated from natural sources.
DNA encoding luciferases is available (see, e.g., SEQ ID NOs. 1-13)
and has been modified (see, e.g., SEQ ID NOs. 3 and 10-13) and
synthetic and alternative substrates have been devised. The DNA
listed herein is only representative of the DNA encoding
luciferases that is available.
[0273] Any bioluminescence generating system, whether synthetic or
isolated form natural sources, such as those set forth in Table 2,
elsewhere herein or known to those of skill in the art, is intended
for use in the combinations, systems and methods provided herein.
Chemiluminescence systems per se, which do not rely on oxygenases
(luciferases ) are not encompassed herein.
[0274] (a) Luciferases
[0275] Luciferases refer to any compound that, in the presence of
any necessary activators, catalyze the oxidation of a
bioluminescence substrate (luciferin) in the presence of molecular
oxygen, whether free or bound, from a lower energy state to a
higher energy state such that the substrate, upon return to the
lower energy state, emits light. For purposes herein, luciferase is
broadly used to encompass enzymes that act catalytically to
generate light by oxidation of a substrate and also photoproteins,
such as aequorin, that act, though not strictly catalytically
(since such proteins are exhausted in the reaction), in conjunction
with a substrate in the presence of oxygen to generate light. These
luciferases, including photoproteins, such as aequorin, are herein
also included among the luciferases. These reagents include the
naturally-occurring luciferases (including photoproteins), proteins
produced by recombinant DNA, and mutated or modified variants
thereof that retain the ability to generate light in the presence
of an appropriate substrate, co-factors and activators or any other
such protein that acts as a catalyst to oxidize a substrate,
whereby light is produced.
[0276] Generically, the protein that catalyzes or initiates the
bioluminescent reaction is referred to as a luciferase, and the
oxidizable substrate is referred to as a luciferin. The oxidized
reaction product is termed oxyluciferin, and certain luciferin
precursors are termed etioluciferin. Thus, for purposes herein
bioluminescence encompasses light produced by reactions that are
catalyzed by (in the case of luciferases that act enzymatically) or
initiated by (in the case of the photoproteins, such as aequorin,
that are not regenerated in the reaction) a biological protein or
analog, derivative or mutant thereof.
[0277] For clarity herein, these catalytic proteins are referred to
as luciferases and include enzymes such as the luciferases that
catalyze the oxidation of luciferin, emitting light and releasing
oxyluciferin. Also included among luciferases are photoproteins,
which catalyze the oxidation of luciferin to emit light but are
changed in the reaction and must be reconstituted to be used again.
The luciferases may be naturally occurring or may be modified, such
as by genetic engineering to improve or alter certain properties.
As long as the resulting molecule retains the ability to catalyze
the bioluminescent reaction, it is encompassed herein.
[0278] Any protein that has luciferase activity (a protein that
catalyzes oxidation of a substrate in the presence of molecular
oxygen to produce light as defined herein) may be used herein. The
preferred luciferases are those that are described herein or that
have minor sequence variations. Such minor sequence variations
include, but are not limited to, minor allelic or species
variations and insertions or deletions of residues, particularly
cysteine residues. Suitable conservative substitutions of amino
acids are known to those of skill in this art and may be made
generally without altering the biological activity of the resulting
molecule. Such substitutions are preferably made in accordance with
those set forth in TABLE 1 as described above.
[0279] The luciferases may be obtained commercially, isolated from
natural sources, expressed in host cells using DNA encoding the
luciferase, or obtained in any manner known to those of skill in
the art. For purposes herein, crude extracts obtained by grinding
up selected source organisms may suffice. Since large quantities of
the luciferase may be desired, isolation of the luciferase from
host cells is preferred. DNA for such purposes is widely available
as are modified forms thereof.
[0280] Examples of luciferases include, but are not limited to,
those isolated from the ctenophores Mnemiopsis (mnemiopsin) and
Beroe ovata (berovin), those isolated from the coelenterates
Aequorea (aequorin), Obelia (obelin), Pelagia, the Renilla
luciferase, the luciferases isolated from the mollusca Pholas
(pholasin), the luciferases isolated from fish, such as
Aristostomias, Pachystomias and Poricthys and from the ostracods,
such as Cypridina (also referred to as Vargula). Preferred
luciferases for use herein are the Aequorin protein, Renilla
luciferase and Cypridina (also called Vargula) luciferase (see,
e.g., SEQ ID NOs. 1, 2, and 4-13). Also, preferred are luciferases
which react to produce red and/or near infrared light. These
include luciferases found in species of Aristostomias, such as A.
scintillans, Pachystomias, Malacosteus, such as M. niger.
[0281] (b) Luciferins
[0282] The substrates for the reaction or for inclusion in the
conjugates include any molecule(s) with which the luciferase reacts
to produce light. Such molecules include the naturally-occurring
substrates, modified forms thereof, and synthetic substrates (see,
e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828). Exemplary luciferins
include those described herein, as well as derivatives thereof,
analogs thereof, synthetic substrates, such as dioxetanes (see,
e.g., U.S. Pat. Nos. 5,004,565 and 5,455,357), and other compounds
that are oxidized by a luciferase in a light-producing reaction
(see, e.g., U.S. Pat. Nos. 5,374,534, 5,098,828 and 4,950,588).
Such substrates also may be identified empirically by selecting
compounds that are oxidized in bioluminescent reactions.
[0283] (c) Activators
[0284] The bioluminescent generating systems also require
additional components discussed herein and known to those of skill
in the art. All bioluminescent reactions require molecular oxygen
in the form of dissolved or bound oxygen. Thus, molecular oxygen,
dissolved in water or in air or bound to a photoprotein, is the
activator for bioluminescence reactions. Depending upon the form of
the components, other activators include, but are not limited to,
ATP (for firefly luciferase), flavin reductase (bacterial systems)
for regenerating FMNH.sub.2 from FMN, and Ca.sup.2+ or other
suitable metal ion (aequorin).
[0285] Most of the systems provided herein will generate light when
the luciferase and luciferin are mixed and exposed to air or water.
The systems that use photoproteins that have bound oxygen, such as
aequorin, however, will require exposure to Ca.sup.2+ (or other
suitable metal ion), which can be provided in the form of an
aqueous composition of a calcium salt. In these instances, addition
of a Ca.sup.2+ (or other suitable metal ion) to a mixture of
luciferase (aequorin) and luciferin (such as coelenterazine) will
result in generation of light. The Renilla system and other
Anthozoa systems also require Ca.sup.2+ (or other suitable metal
ion).
[0286] If crude preparations are used, such as ground up Cypridina
(shrimp) or ground fireflies, it may be necessary to add only
water. In instances in which fireflies (or a firefly or beetle
luciferase) are used the reaction may only require addition ATP.
The precise components will be apparent, in light of the disclosure
herein, to those of skill in this art or may be readily determined
empirically.
[0287] It is also understood that these mixtures will also contain
any additional salts or buffers or ions that are necessary for each
reaction to proceed. Since these reactions are well-characterized,
those of skill in the art will be able to determine precise
proportions and requisite components. Selection of components will
depend upon the apparatus, article of manufacture and luciferase.
Various embodiments are described and exemplified herein; in view
of such description, other embodiments will be apparent.
[0288] (d) Reactions
[0289] In all embodiments, all but one component, either the
luciferase or luciferin, of a bioluminescence generating system
will be mixed or packaged with or otherwise combined. Since the
result to be achieved is the production of light visible to the
naked eye for qualitative, not quantitative, diagnostic purposes,
the precise proportions and amounts of components of the
bioluminescence reaction need not be stringently determined or met.
They must be sufficient to produce light. Generally, an amount of
luciferin and luciferase sufficient to generate a visible glow is
used; this amount can be readily determined empirically and is
dependent upon the selected system and selected application. Where
quantitative measurements are required, more precision may be
required.
[0290] For purposes herein, such amount is preferably at least the
concentrations and proportions used for analytical purposes by
those of skill in the such arts. Higher concentrations may be used
if the glow is not sufficiently bright. Alternatively, a
microcarrier coupled to more than one luciferase molecule linked to
a targeting agent may be utilized to increase signal output. Also
because the conditions in which the reactions are used are not
laboratory conditions and the components are subject to storage,
higher concentration may be used to overcome any loss of activity.
Typically, the amounts are 1 mg, preferably 10 mg and more
preferably 100 mg, of a luciferase per liter of reaction mixture or
1 mg, preferably 10 mg, more preferably 100 mg. Compositions may
contain at least about 0.01 mg/l, and typically 0.1 mg/l, 1 mg/l,
10 mg/l or more of each component on the item. The amount of
luciferin is also between about 0.01 and 100 mg/l, preferably
between 0.1 and 10 mg/l, additional luciferin can be added to many
of the reactions to continue the reaction. In embodiments in which
the luciferase acts catalytically and does not need to be
regenerated, lower amounts of luciferase can be used. In those in
which it is changed during the reaction, it also can be
replenished; typically higher concentrations will be selected.
Ranges of concentration per liter (or the amount of coating on
substrate the results from contacting with such composition) of
each component on the order of 0.1 to 20 mg, preferably 0.1 to 10
mg, more preferably between about 1 and 10 mg of each component
will be sufficient. When preparing coated substrates, as described
herein, greater amounts of coating compositions containing higher
concentrations of the luciferase or luciferin may be used.
[0291] Thus, for example, in presence of calcium, 5 mg of
luciferin, such as coelenterazine, in one liter of water will glow
brightly for at least about 10 to 20 minutes, depending on the
temperature of the water, when about 10 mg of luciferase such as
aequorin photoprotein luciferase or luciferase from Renilla, is
added thereto. Increasing the concentration of luciferase, for
example, to 100 mg/l, provides a particularly brilliant display of
light.
[0292] It is understood, that concentrations and amounts to be used
depend upon the selected bioluminescence generating system but
these may be readily determined empirically. Proportions,
particularly those used when commencing an empirical determination,
are generally those used for analytical purposes, and amounts or
concentrations are at least those used for analytical purposes, but
the amounts can be increased, particularly if a sustained and
brighter glow is desired.
[0293] For purposes herein, Renilla reniformis GFP is added to the
reaction in order to shift the spectrum of the generated light.
[0294] 2. The Renilla System
[0295] Renilla, also known as soft coral sea pansies, are members
of the class of coelenterates Anthozoa, which includes other
bioluminescent genera, such as Cavarnularia, Ptilosarcus,
Stylatula, Acanthoptilum, and Parazoanthus. Bioluminescent members
of the Anthozoa genera contain luciferases and luciferins that are
similar in structure (see, e.g., Cormier et al. (1973) J. Cell.
Physiol. 81:291-298; see, also Ward et al. (1975) Proc. Natl. Acad.
Sci. U.S.A. 72:2530-2534). The luciferases and luciferins from each
of these anthozoans crossreact with one another and produce a
characteristic blue luminescence.
[0296] Renilla luciferase and the other coelenterate and ctenophore
luciferases, such as the aequorin photoprotein, use imidazopyrazine
substrates, particularly the substrates generically called
coelenterazine (see, formulae (I) and (II) of Section C.4.b,
below). Other genera that have luciferases that use a
coelenterazine include: squid, such as Chiroteuthis, Eucleoteuthis,
Onychoteuthis, Watasenia, cuttlefish, Sepiolina; shrimp, such as
Oplophorus, Acanthophyra, Sergestes, and Gnathophausia; deep-sea
fish, such as Argyropelecus, Yarella, Diaphus, Gonadostomias and
Neoscopelus.
[0297] Renilla luciferase does not, however, have bound oxygen, and
thus requires dissolved oxygen in order to produce light in the
presence of a suitable luciferin substrate. Since Renilla
luciferase acts as a true enzyme (i.e., it does not have to be
reconstituted for further use) the resulting luminescence can be
long-lasting in the presence of saturating levels of luciferin.
Also, Renilla luciferase is relatively stable to heat.
[0298] Renilla luciferases, DNA encoding Renilla reniformis
luciferase, and use of the Renilla reniformis DNA to produce
recombinant luciferase, as well as DNA encoding luciferase from
other coelenterates, are well known and available (see, e.g., SEQ
ID NO. 1, U.S. Pat. Nos. 5,418,155 and 5,292,658; see, also,
Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126:1259-1268;
Cormier (1981) "Renilla and Aequorea bioluminescence" in
Bioluminescence and Chemiluminescence, pp. 225-233; Charbonneau et
al. (1979) J. Biol. Chem. 254:769-780; Ward et al. (1979) J. Biol.
Chem. 254:781-788; Lorenz et al. (1981) Proc. Natl. Acad, Sci.
U.S.A. 88: 4438-4442; Hori et al. (1977) Proc. Natl. Acad. Sci.
U.S.A. 74:4285-4287; Hori et al. (1975) Biochemistry 14:2371-2376;
Hori et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287;
Inouye et al. (1975) Jap. Soc. Chem. Lett. 141-144; and Matthews et
al. (1979) Biochemistry 16:85-91). The DNA encoding Renilla
reniformis luciferase and host cells containing such DNA provide a
convenient means for producing large quantities of Renilla
reniformis enzyme, such as in those known to those of skill in the
art (see, e.g., U.S. Pat. Nos. 5,418,155 and 5,292,658, which
describe recombinant production of Renilla reniformis
luciferase).
[0299] When used herein, the Renilla luciferase can be packaged in
lyophilized form, encapsulated in a vehicle, either by itself or in
combination with the luciferin substrate. Prior to use the mixture
is contacted with an aqueous composition, preferably a phosphate
buffered saline pH 7-8; dissolved O.sub.2 will activate the
reaction. Final concentrations of luciferase in the glowing mixture
will be on the order of 0.01 to 1 mg/l or more. Concentrations of
luciferin will be at least about 10.sup.-8 M, but 1 to 100 or more
orders of magnitude higher to produce a long lasting
bioluminescence.
[0300] In certain embodiments herein, about 1 to 10 mg, or
preferably 2-5 mg, more preferably about 3 mg of coelenterazine
will be used with about 100 mg of Renilla luciferase. The precise
amounts, of course can be determined empirically, and, also will
depend to some extent on the ultimate concentration application. In
particular, addition of about 0.25 ml of a crude extract from the
bacteria that express Renilla to 100 ml of a suitable assay buffer
and about 0.005 .mu.g was sufficient to produce a visible and
lasting glow (see, U.S. Pat. Nos. 5,418,155 and 5,292,658, which
describe recombinant production of Renilla reniformis
luciferase).
[0301] Lyophilized mixtures, and compositions containing the
Renilla luciferase are also provided. The luciferase or mixtures of
the luciferase and luciferin may also be encapsulated into a
suitable delivery vehicle, such as a liposome, glass particle,
capillary tube, drug delivery vehicle, gelatin, time release
coating or other such vehicle. The luciferase may also be linked to
a substrate, such as biocompatible materials.
[0302] 3. Ctenophore Systems
[0303] Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata
(berovin), and coelenterates, such as Aequorea (aequorin), Obelia
(obelin) and Pelagia, produce bioluminescent light using similar
chemistries (see, e.g., Stephenson et al. (1981) Biochimica et
Biophysica Acta 678:65-75; Hart et al. (1979) Biochemistry
18:2204-2210; International PCT Application No. WO 94/18342, which
is based on U.S. application Ser. No. 08/017,116, U.S. Pat. No.
5,486,455 and other references and patents cited herein). The
Aequorin and Renilla systems are representative and are described
in detail herein as exemplary and as among the presently preferred
systems. The Aequorin and Renilla systems can use the same
luciferin and produce light using the same chemistry, but each
luciferase is different. The Aequorin luciferase aequorin, as well
as, for example, the luciferases mnemiopsin and berovin, is a
photoprotein that includes bound oxygen and bound luciferin,
requires Ca.sup.2+ (or other suitable metal ion) to trigger the
reaction, and must be regenerated for repeated use; whereas, the
Renilla luciferase acts as a true enzyme because it is unchanged
during the reaction and it requires dissolved molecular oxygen.
[0304] 4. The Aequorin System
[0305] The aequorin system is well known (see, e.g., Tsuji et al.
(1986) "Site-specific mutagenesis of the calcium-binding
photoprotein aequorin," Proc. Natl. Acad. Sci. USA 83:8107-8111;
Prasher et al. (1985) "Cloning and Expression of the cDNA Coding
for Aequorin, a Bioluminescent Calcium-Binding Protein,"
Biochemical and Biophysical Research Communications 126:1259-1268;
Prasher et al. (1986) Methods in Enzymology 133:288-297; Prasher,
et al. (1987) "Sequence Comparisons of cDNAs Encoding for Aequorin
Isotypes," Biochemistry 26:1326-1332; Charbonneau et al. (1985)
"Amino Acid Sequence of the Calcium-Dependent Photoprotein
Aequorin, " Biochemistry 24:6762-6771; Shimomura et al. (1981)
"Resistivity to denaturation of the apoprotein of aequorin and
reconstitution of the luminescent photoprotein from the partially
denatured apoprotein," Biochem. J. 199:825-828; Inouye et al.
(1989) J. Biochem. 105:473-477; Inouye et al. (1986) "Expression of
Apoacquorin Complementary DNA in Escherichia coli, " Biochemistry
25:8425-8429; Inouye et al. (1985) "Cloning and sequence analysis
of cDNA for the luminescent protein aequorin," Proc. Natl. Acad.
Sci. USA 82:3154-3158; Prendergast, et al. (1978) "Chemical and
Physical Properties of Aequorin and the Green Fluorescent Protein
Isolated from Aequorea forskalea" J. Am. Chem. Soc. 17:3448-3453;
European patent application 0 540 064 Al; European patent
application 0 226 979 A2, European patent application 0 245 093 A1
and European patent application 0 245 093 B1; U.S. Pat. No.
5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S.
Pat. No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No.
5,162,227; and SEQ ID Nos. 5-13, which set forth DNA encoding the
apoprotein; and a form, described in U.S. Pat. No. 5,162,227,
European patent application 0 540 064 A1 and Sealite Sciences
Technical Report No. 3 (1994), is commercially available from
Sealite, Sciences, Bogart, Ga. as AQUALITE.RTM.).
[0306] This system is among the preferred systems for use herein.
As will be evident, since the aequorin photoprotein includes
noncovalently bound luciferin and molecular oxygen, it is suitable
for storage in this form as a lyophilized powder or encapsulated
into a selected delivery vehicle. The system can be encapsulated
into pellets, such as liposomes or other delivery vehicles. When
used, the vehicles are contacted with a composition, even tap
water, that contains Ca.sup.2+ (or other suitable metal ion), to
produce a mixture that glows.
[0307] a. Aequorin and Related Photoproteins
[0308] The photoprotein, aequorin, isolated from the jellyfish,
Aequorea, emits light upon the addition of Ca.sup.2+ (or other
suitable metal ion). The aequorin photoprotein, which includes
bound luciferin and bound oxygen that is released by Ca.sup.2+ ,
does not require dissolved oxygen. Luminescence is triggered by
calcium, which releases oxygen and the luciferin substrate
producing apoaqueorin.
[0309] The bioluminescence photoprotein aequorin is isolated from a
number of species of the jellyfish Aequorea. It is a 22 kilodalton
(kD) molecular weight peptide complex (see, e.g., Shimomura et al.
(1962) J. Cellular and Comp. Physiol. 59:233-238; Shimomura et al.
(1969) Biochemistry 8:3991-3997; Kohama et al. (1971) Biochemistry
10:4149-4152; and Shimomura et al. (1972) Biochemistry
11:1602-1608). The native protein contains oxygen and a
heterocyclic compound coelenterazine, a luciferin, (see, below)
noncovalently bound thereto. The protein contains three calcium
binding sites. Upon addition of trace amounts Ca.sup.2+ (or other
suitable metal ion, such as strontium) to the photoprotein, it
undergoes a conformational change that catalyzes the oxidation of
the bound coelenterazine using the protein-bound oxygen. Energy
from this oxidation is released as a flash of blue light, centered
at 469 nm. Concentrations of calcium ions as low as 10.sup.-6 M are
sufficient to trigger the oxidation reaction.
[0310] Naturally-occurring apoaequorin is not a single compound but
rather is a mixture of microheterogeneous molecular species.
Aequoria jellyfish extracts contain as many as twelve distinct
variants of the protein (see, e.g., Prasher et al. (187)
Biochemistry 26:1326-1332; Blinks et al. (1975) Fed. Proc. 34:474).
DNA encoding numerous forms has been isolated (see, e.g., SEQ ID
NOs. 5-9 and 13).
[0311] The photoprotein can be reconstituted (see, e.g., U.S. Pat.
No. 5,023,181) by combining the apoprotein, such as a protein
recombinantly produced in E. coli, with a coelenterazine, such as a
synthetic coelenterazine, in the presence of oxygen and a reducing
agent (see, e.g., Shimomura et al. (1975) Nature 256:236-238;
Shimomura et al. (1981) Biochemistry J. 199:825-828), such as
2-mercaptoethanol, and also EDTA or EGTA (concentrations between
about 5 to about 100 mM or higher for applications herein) tie up
any Ca.sup.2+ to prevent triggering the oxidation reaction until
desired. DNA encoding a modified form of the apoprotein that does
not require 2-mercaptoethanol for reconstitution is also available
(see, e.g., U.S. Pat. No. U.S. Pat. No. 5,093,240). The
reconstituted photoprotein is also commercially available (sold,
e.g., under the trademark AQUALITE.RTM., which is described in U.S.
Pat. No. 5,162,227).
[0312] The light reaction is triggered by adding Ca.sup.2+ at a
concentration sufficient to overcome the effects of the chelator
and achieve the 10.sup.-6 M concentration. Because such low
concentrations of Ca.sup.2+ can trigger the reaction, for use in
the methods herein, higher concentrations of chelator may be
included in the compositions of photoprotein. Accordingly, higher
concentrations of added Ca.sup.2+ in the form of a calcium salt
will be required. Precise amounts may be empirically determined.
For use herein, it may be sufficient to merely add water to the
photoprotein, which is provided in the form of a concentrated
composition or in lyophilized or powdered form. Thus, for purposes
herein, addition of small quantities of Ca.sup.2+, such as those
present in phosphate buffered saline (PBS) or other suitable
buffers or the moisture on the tissue to which the compositions are
contacted, should trigger the bioluminescence reaction.
[0313] Numerous isoforms of the aequorin apoprotein been identified
isolated. DNA encoding these proteins has been cloned, and the
proteins and modified forms thereof have been produced using
suitable host cells (see, e.g., U.S. Pat. Nos. 5,162,227,
5,360,728, 5,093,240; see, also, Prasher et al. (1985) Biophys.
Biochem. Res. Commun. 126:1259-1268; Inouye et al. (1986)
Biochemistry 25:8425-8429). U.S. Pat. No. 5,093,240; U.S. Pat. No.
5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,288,623; U.S.
Pat. No. 5,422,266, U.S. Pat. No. 5,162,227 and SEQ ID Nos. 5-13,
which set forth DNA encoding the apoprotein; and a form is
commercially available form Sealite, Sciences, Bogart, Ga. as
AQUALITE.RTM.). DNA encoding apoaequorin or variants thereof is
useful for recombinant production of high quantities of the
apoprotein. The photoprotein is reconstituted upon addition of the
luciferin, coelenterazine, preferably a sulfated derivative
thereof, or an analog thereof, and molecular oxygen (see, e.g.,
U.S. Pat. No. 5,023,181). The apoprotein and other constituents of
the photoprotein and bioluminescence generating reaction can be
mixed under appropriate conditions to regenerate the photoprotein
and concomitantly have the photoprotein produce light.
Reconstitution requires the presence of a reducing agent, such as
mercaptoethanol, except for modified forms, discussed below, that
are designed so that a reducing agent is not required (see, e.g.,
U.S. Pat. No. 5,093,240).
[0314] For use herein, it is preferred aequorin is produced using
DNA, such as that set forth in SEQ ID NOs. 5-13 and known to those
of skill in the art or modified forms thereof. The DNA encoding
aequorin is expressed in a host cell, such as E. coli, isolated and
reconstituted to produce the photoprotein (see, e.g., U.S. Pat.
Nos. 5,418,155, 5,292,658, 5,360,728, 5,422,266, 5,162,227).
[0315] Of interest herein, are forms of the apoprotein that have
been modified so that the bioluminescent activity is greater than
unmodified apoaequorin (see, e.g., U.S. Pat. No. 5,360,728, SEQ ID
NOs. 10-12). Modified forms that exhibit greater bioluminescent
activity than unmodified apoaequorin include proteins including
sequences set forth in SEQ ID NOs. 10-12, in which aspartate 124 is
changed to serine, glutamate 135 is changed to serine, and glycine
129 is changed to alanine, respectively. Other modified forms with
increased bioluminescence are also available.
[0316] For use in certain embodiments herein, the apoprotein and
other components of the aequorin bioluminescence generating system
are packaged or provided as a mixture, which, when desired is
subjected to conditions under which the photoprotein reconstitutes
from the apoprotein, luciferin and oxygen (see, e.g., U.S. Pat. No.
5,023,181; and U.S. Pat. No. 5,093,240). Particularly preferred are
forms of the apoprotein that do not require a reducing agent, such
as 2-mercapto-ethanol, for reconstitution. These forms, described,
for example in U.S. Pat. No. 5,093,240 (see, also Tsuji et al.
(1986) Proc. Natl. Acad. Sci. U.S.A. 83:8107-8111), are modified by
replacement of one or more, preferably all three cysteine residues
with, for example serine. Replacement may be effected by
modification of the DNA encoding the aequorin apoprotein, such as
that set forth in SEQ ID NO. 5, and replacing the cysteine codons
with serine.
[0317] The photoproteins and luciferases from related species, such
as Obelia are also contemplated for use herein. DNA encoding the
Ca.sup.2+-activated photoprotein obelin from the hydroid polyp
Obelia longissima is known and available (see, e.g., Illarionov et
al. (1995) Gene 153:273-274; and Bondar et al. (1995) Biochim.
Biophys. Acta 1231:29-32). This photoprotein can also be activated
by Mn.sup.2+ (see, e.g., Vysotski et al. (1995) Arch. Bioch.
Biophys. 316:92-93, Vysotski et al. (1993) J. Biolumin. Chemilumin.
8:301-305).
[0318] In general for use herein, the components of the
bioluminescence are packaged or provided so that there is
insufficient metal ions to trigger the reaction. When used, the
trace amounts of triggering metal ion, particularly Ca.sup.2+ is
contacted with the other components. For a more sustained glow,
aequorin can be continuously reconstituted or can be added or can
be provided in high excess.
[0319] b. Luciferin
[0320] The aequorin luciferin is coelenterazine and analogs
therein, which include molecules including the structure (formula
(I)): 1
[0321] in which R.sub.1 is CH.sub.2C.sub.6H.sub.5 or CH.sub.3;
R.sub.2 is C.sub.6H.sub.5, and R.sub.3 is p-C.sub.6H.sub.4OH or
CH.sub.3 or other such analogs that have activity. Preferred
coelenterazine has the structure in which R.sup.1 is
p-CH.sub.2C.sub.6H.sub.4OH, R.sub.2 is C.sub.6H.sub.5, and R.sub.3
is p-C.sub.6H.sub.4OH, which can be prepared by known methods (see,
e.g., Inouye et al. (1975) Jap. Chem. Soc., Chemistry Lttrs. pp
141-144; and Hart et al. (1979) Biochemistry 18:2204-2210). Among
the preferred analogs, are those that are modified, whereby the
spectral frequency of the resulting light is shifted to another
frequency.
[0322] The preferred coelenterazine has the structure (formula
(II)): 2
[0323] and sulfated derivatives thereof
[0324] Another coelentratrazine has formula (V): 3
[0325] (see, Hart et al. (1979) Biochemistry 18:2204-2210). Using
this derivative in the presence of luciferase all of the light is
in the ultraviolet with a peak at 390 nm. Upon addition of GFP, all
light emitted is now in the visible range with a peak at 509 nm
accompanied by an about 200-fold increase in the amount of light
emitted. Viewed with a cut-off filter of 470 nm, in the light yield
in the absence of GFP would be about zero, and would be detectable
in the presence of GFP. This provides the basis for an immunoassay
described in the EXAMPLES.
[0326] The reaction of coelenterazine when bound to the aequorin
photoprotein with bound oxygen and in the presence of Ca.sup.2+ can
represented as follows: 4
[0327] The photoprotein aequorin (which contains apoaequorin bound
to a coelenterate luciferin molecule) and Renilla luciferase,
discussed below, can use the same coelenterate luciferin. The
aequorin photoprotein catalyses the oxidation of coelenterate
luciferin (coelenterazine) to oxyluciferin (coelenteramide) with
the concomitant production of blue light (lambda.sub.max=469
nm).
[0328] Importantly, the sulfate derivative of the coelenterate
luciferin (lauryl-luciferin) is particularly stable in water, and
thus may be used in a coelenterate-like bioluminescent system. In
this system, adenosine diphosphate (ADP) and a sulpha-kinase are
used to convert the coelenterazine to the sulphated form. Sulfatase
is then used to reconvert the lauryl-luciferin to the native
coelenterazine. Thus, the more stable lauryl-luciferin is used in
the item to be illuminated and the luciferase combined with the
sulfatase are added to the luciferin mixture when illumination is
desired.
[0329] Thus, the bioluminescent system of Aequorea is particularly
suitable for use in the methods herein. The particular amounts and
the manner in which the components are provided depends upon the
type of neoplasia or specialty tissue to be visualized. This system
can be provided in lyophilized form, that will glow upon addition
of Ca.sup.2+ . It can be encapsulated, linked to microcarriers,
such as microbeads, or in as a compositions, such as a solution or
suspension, preferably in the presence of sufficient chelating
agent to prevent triggering the reaction. The concentration of the
aequorin photoprotein will vary and can be determined empirically.
Typically concentrations of at least 0.1 mg/l, more preferably at
least 1 mg/l and higher, will be selected. In certain embodiments,
1-10 mg luciferin/100 mg of luciferase will be used in selected
volumes and at the desired concentrations will be used.
[0330] 5. Crustacean, Particularly Cyrpidina Systems
[0331] The ostracods, such as Vargula serratta, hilgendorfil and
noctiluca are small marine crustaceans, sometimes called sea
fireflies. These sea fireflies are found in the waters off the
coast of Japan and emit light by squirting luciferin and luciferase
into the water, where the reaction, which produces a bright blue
luminous cloud, occurs. The reaction involves only luciferin,
luciferase and molecular oxygen, and, thus, is very suitable for
application herein.
[0332] The systems, such as the Vargula bioluminescent systems, are
particularly preferred herein because the components are stable at
room temperature if dried and powdered and will continue to react
even if contaminated. Further, the bioluminescent reaction requires
only the luciferin/luciferase components in concentrations as low
as 1:40 parts per billion to 1:100 parts per billion, water and
molecular oxygen to proceed. An exhausted system can be renewed by
addition of luciferin.
[0333] a. Vargula Luciferase
[0334] The Vargula luciferase is water soluble and is among those
preferred for use in the methods herein. Vargula luciferase is a
555-amino acid polypeptide that has been produced by isolation from
Vargula and also using recombinant technology by expressing the DNA
in suitable bacterial and mammalian host cells (see, e.g., Thompson
et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571; Inouye et
al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:9584-9587; Johnson et
al. (1978) Methods in Enzymology LVII:331-349; Tsuji et al. (1978)
Methods Enzymol. 57:364-72; Tsuji (1974) Biochemistry 13:5204-5209;
Japanese patent application No. JP 3-30678 Osaka; and European
patent application No. EP 0 387 355 A1).
[0335] (1) Purification from Cypridina
[0336] Methods for purification of Vargula (Cypridina) luciferase
are well known. For example, crude extracts containing the active
can be readily prepared by grinding up or crushing the Vargula
shrimp. In other embodiments, a preparation of Cypridina
hilgendorfi luciferase can be prepared by immersing stored frozen
C. hilgendorfi in distilled water containing, 0.5-5.0 M salt,
preferably 0.5-2.0 M sodium or potassium chloride, ammonium
sulfate, at 0-30.degree. C., preferably 0-10.degree. C., for 1-48
hr, preferably 10-24 hr, for extraction followed by hydrophobic
chromatography and then ion exchange or affinity chromatography
(TORAY IND INC, Japanese patent application JP 4258288, published
Sep. 14, 1993; see, also, Tsuji et al. (1978) Methods Enzymol.
57:364-72 for other methods).
[0337] (2) Preparation by Recombinant Methods
[0338] The luciferase is preferably produced by expression of
cloned DNA encoding the luciferase (European patent application No.
0 387 355 A1; International PCT Application No. WO 95/001542; see,
also SEQ ID No. 5, which sets forth the sequence from Japanese
patent application No. JP 3-30678 and Thompson et al. (1989) Proc.
Natl. Acad. Sci. U.S.A. 86:6567-6571) DNA encoding the luciferase
or variants thereof is introduced into E. coli using appropriate
vectors and isolated using standard methods.
[0339] b. Vargula Luciferin
[0340] The natural luciferin is a substituted imidazopyrazine
nucleus, such a compound of formula (III): 5
[0341] The luciferin can be isolated from ground dried Vargula by
heating the extract, which destroys the luciferase but leaves the
luciferin intact (see, e.g., U.S. Pat. No. 4,853,327).
[0342] Analogs thereof and other compounds that react with the
luciferase in a light producing reaction also may be used.
[0343] Other bioluminescent organisms that have luciferases that
can react with the Vargula luciferin include, the genera Apogon,
Parapriacanthus and Porichthys.
[0344] c. Reaction
[0345] The luciferin upon reaction with oxygen forms a dioxetanone
intermediate (which includes a cyclic peroxide similar to the
firefly cyclic peroxide molecule intermediate). In the final step
of the bioluminescent reaction, the peroxide breaks down to form
CO.sub.2 and an excited carbonyl. The excited molecule then emits a
blue to blue-green light.
[0346] The optimum pH for the reaction is about 7. For purposes
herein, any pH at which the reaction occurs may be used. The
concentrations of reagents are those normally used for analytical
reactions or higher (see, e.g., Thompson et al. (1990) Gene
96:257-262). Typically concentrations of the luciferase between 0.1
and 10 mg/l, preferably 0.5 to 2.5 mg/l will be used. Similar
concentrations or higher concentrations of the luciferin may be
used.
[0347] 6. Insect Bioluminescent Systems Including Fireflies, Click
Beetles, and Other Insect System
[0348] The biochemistry of firefly bioluminescence was the first
bioluminescent system to be characterized (see, e.g., Wienhausen et
al. (1985) Photochemistry and Photobiology 42:609-611; McElroy et
al. (1966) in Molecular Architecture in cell Physiology, Hayashi et
al., eds. Prentice Hall, Inc., Englewood Cliffs, N.J., pp. 63-80)
and it is commercially available (e.g., from Promega Corporation,
Madison, Wis., see, e.g., Leach et al. (1986) Methods in Enzymology
133:51-70, esp. Table 1). Luciferases from different species of
fireflies are antigenically similar. These species include members
of the genera Photinus, Photurins and Luciola. Further, the
bioluminescent reaction produces more light at 30.degree. C. than
at 20.degree. C., the luciferase is stabilized by small quantities
of bovine albumin serum, and the reaction can be buffered by
tricine.
[0349] a. Luciferase
[0350] DNA clones encoding luciferases from various insects and the
use to produce the encoded luciferase is well known. For example,
DNA clones that encode luciferase from Photinus pyralis, Luciola
cruciata (see, e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci.
U.S.A. 82:7870-7873; de We et al. (1986) Methods in Enzymology
133:3; U.S. Pat. No. 4,968,613, see, also SEQ ID NO. 3) are
available. The DNA has also been expressed in Saccharomyces (see,
e.g., Japanese Application No. JP 63317079, published Dec. 26,
1988, KIKKOMAN CORP) and in tobacco.
[0351] In addition to the wild-type luciferase modified insect
luciferases have been prepared. For example, heat stable luciferase
mutants, DNA-encoding the mutants, vectors and transformed cells
for producing the luciferases are available. A protein with 60%
amino acid sequence homology with luciferases from Photinus
pyralis, Luciola mingrelica, L. cruciata or L. lateralis and having
luciferase activity is available (see, e.g., International PCT
Application No. WO 95/25798). It is more stable above 30.degree. C.
than naturally-occurring insect luciferases and may also be
produced at 37.degree. C. or above, with higher yield.
[0352] Modified luciferases can generate light at different
wavelengths (compared with native luciferase), and thus, may be
selected for their color-producing characteristics. For example,
synthetic mutant beetle luciferase(s) and DNA encoding such
luciferases that produce bioluminescence at a wavelength different
from wild-type luciferase are known (Promega Corp, International
PCT Application No. WO 95/18853, which is based on U.S. application
Ser. No. 08/177,081). The mutant beetle luciferase has an amino
acid sequence differing from that of the corresponding wild-type
Luciola cruciata (see, e.g., U.S. Pat. Nos. 5,182,202, 5,219,737,
5,352,598, see, also SEQ ID No.3) by a substitution(s) at one or
two positions. The mutant luciferase produces a bioluminescence
with a wavelength of peak intensity that differs by at least 1 nm
from that produced by wild-type luciferases.
[0353] Other mutant luciferases can be produced. Mutant luciferases
with the amino acid sequence of wild-type luciferase, but with at
least one mutation in which valine is replaced by isoleucine at the
amino acid number 233, valine by isoleucine at 239, serine by
asparagine at 286, glycine by serine at 326, histidine by tyrosine
at 433 or proline by serine at 452 are known (see, e.g., U.S. Pat.
Nos. 5,219,737, and 5,330,906). The luciferases are produced by
expressing DNA-encoding each mutant luciferase in E. coli and
isolating the protein. These luciferases produce light with colors
that differ from wild-type. The mutant luciferases catalyze
luciferin to produce red (.lambda. 609 nm and 612 nm), orange (k
595 and 607 nm) or green (.lambda. 558 nm) light. The other
physical and chemical properties of mutant luciferase are
substantially identical to native wild type-luciferase. The mutant
luciferase has the amino acid sequence of Luciola cruciata
luciferase with an alteration selected from Ser 286 replaced by
Asn, Gly 326 replaced by Ser, His 433 replaced by Tyr or Pro 452
replaced by Ser. Thermostable luciferases are also available (see,
e.g., U.S. Pat. No. 5,229,285; see, also International PCT
Application No. WO 95/25798, which provides Photinus luciferase in
which the glutamate at position 354 is replaced with lysine and
Luciola luciferase in which the glutamate at 356 is replaced with
lysine).
[0354] These mutant luciferases as well as the wild type
luciferases can be used in combination with the GFPs provided
herein particularly in instances when a variety of colors are
desired or when stability at higher temperatures is desired.
[0355] b. Luciferin
[0356] The firefly luciferin is a benzothiazole: 6
[0357] Analogs of this luciferin and synthetic firefly luciferins
are also known to those of skill in art (see, e.g., U.S. Pat. No.
5,374,534 and 5,098,828). These include compounds of formula (IV)
(see, U.S. Pat. No. 5,098,828): 7
[0358] in which:
[0359] R.sup.1 is hydroxy, amino, linear or branched
C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkyenyloxy, an L-amino
acid radical bond via the .alpha.-amino group, an oligopeptide
radical with up to ten L-amino acid units linked via the
.alpha.-amino group of the terminal unit;
[0360] R.sup.2 is hydrogen, H.sub.2PO.sub.3, HSO.sub.3,
unsubstituted or phenyl substituted linear or branched
C.sub.1-C.sub.20 alkyl or C.sub.2-C.sub.20 alkenyl, aryl containing
6 to 18 carbon atoms, or R.sup.3--C(O)--; and
[0361] R.sup.3is an unsubstituted or phenyl substituted linear or
branched C.sub.1-C.sub.20 alkyl or C.sub.2-C.sub.20 alkenyl, aryl
containing 6 to 18 carbon atoms, a nucleotide radical with 1 to 3
phosphate groups, or a glycosidically attached mono- or
disaccharide, except when formula (IV) is a D-luciferin or
D-luciferin methyl ester.
[0362] Modified luciferins that have been modified to produce light
of shifted frequencies are known to those of skill in the art.
[0363] c. Reaction
[0364] The reaction catalyzed by firefly luciferases and related
insect luciferases requires ATP, Mg.sup.2+ as well as molecular
oxygen. Luciferin must be added exogenously. Firefly luciferase
catalyzes the firefly luciferin activation and the subsequent steps
leading to the excited product. The luciferin reacts with ATP to
form a luciferyl adenylate intermediate. This intermediate then
reacts with oxygen to form a cyclic luciferyl peroxy species,
similar to that of the coelenterate intermediate cyclic peroxide,
which breaks down to yield CO.sub.2 and an excited state of the
carbonyl product. The excited molecule then emits a yellow light;
the color, however, is a function of pH. As the pH is lowered the
color of the bioluminescence changes from yellow-green to red.
[0365] Different species of fireflies emit different colors of
bioluminescence so that the color of the reaction will be dependent
upon the species from which the luciferase is obtained.
Additionally, the reaction is optimized at pH 7.8.
[0366] Addition of ATP and luciferin to a reaction that is
exhausted produces additional light emission. Thus, the system,
once established, is relatively easily maintained. Therefore, it is
highly suitable for use herein in embodiments in which a sustained
glow is desired.
[0367] 7. Other Systems
[0368] Numerous other systems are known and have been described in
detail for example in U.S. Pat. Nos. 5,876,995, 6,152,358 and
6,113,886).
[0369] a. Bacterial Systems
[0370] Luminous bacteria typically emit a continuous light, usually
blue-green. When strongly expressed, a single bacterium may emit
10.sup.4 to 10.sup.5 photons per second. Bacterial bioluminescence
systems include, among others, those systems found in the
bioluminescent species of the genera Photobacterium, Vibrio and
Xenorhabdus. These systems are well known and well characterized
(see, e.g., Baldwin et al. (1984) Biochemistry 23:3663-3667; Nicoli
et al. (1974) J. Biol. Chem. 249:2393-2396; Welches et al. (1981)
Biochemistry 20:512-517; Engebrecht et al. (1986) Methods in
Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology
172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70;
U.S. Pat. No. 4,581,335).
[0371] (1) Luciferases
[0372] Bacterial luciferase, as exemplified by luciferase derived
from Vibrio harveyi (EC 1.14.14.3, alkanol reduced-FMN-oxygen
oxidoreductase 1-hydroxylating, luminescing), is a mixed function
oxidase, formed by the association of two different protein
subunits .alpha. and .beta.. The .alpha.-subunit has an apparent
molecular weight of approximately 42,000 kDa and the .beta.-subunit
has an apparent molecular weight of approximately 37,000 kDa (see,
e.g., Cohn et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 90:102-123).
These subunits associate to form a 2-chain complex luciferase
enzyme, which catalyzes the light emitting reaction of
bioluminescent bacteria, such as Vibrio harveyi (U. S. Pat. No.
4,581,335; Belas et al. (1982) Science 218:791-793), Vibrio
fischeri (Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et
al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158) and other
marine bacteria.
[0373] Bacterial luciferase genes have been cloned (see, e.g., U.S.
Pat. No. 5,221,623; U.S. Pat. No. 4,581,335; European patent
application No. EP 386 691 A). Plasmids for expression of bacterial
luciferase, such as Vibrio harveyi, include pFIT001 (NRRL B-18080),
pPALE001 (NRRL B-18082) and pMR19 (NRRL B-18081)) are known. For
example the sequence of the entire lux regulon from Vibiro fisheri
has been determined (Baldwin et al. (1984), Biochemistry
23:3663-3667; Baldwin et al. (1981) Biochem. 20:512-517; Baldwin et
al. (1984) Biochem. 23:3663-3667; see, also, e.g., U.S. Pat. Nos.
5,196,318, 5,221,623, and 4,581,335). This regulon includes luxI
gene, which encodes a protein required for autoinducer synthesis
(see, e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci. U.S.A.
81:4154-4158), the luxC, luxD, and luxE genes, which encode enzymes
that provide the luciferase with an aldehyde substrate, and the
luxA and luxB genes, which encode the alpha and beta subunits of
the luciferase.
[0374] Lux genes from other bacteria have also been cloned and are
available (see, e.g., Cohn et al. (1985) J. Biol. Chem.
260:6139-6146; U.S. Pat. No. 5,196,524, which provides a fusion of
the luxA and luxB genes from Vibrio harveyl). Thus, luciferase
alpha and beta subunit-encoding DNA is provided and can be used to
produce the luciferase. DNA encoding the .alpha. (1065 bp) and
.beta. (984 bp) subunits, DNA encoding a luciferase gene of 2124
bp, encoding the alpha and beta subunits, a recombinant vector
containing DNA encoding both subunits and a transformed E. coli and
other bacterial hosts for expression and production of the encoded
luciferase are available. In addition, bacterial luciferases are
commercially available.
[0375] (2) Luciferins
[0376] Bacterial luciferins include: 8
[0377] in which the tetradecanal with reduced flavin mononucleotide
are considered luciferin since both are oxidized during the light
emitting reaction.
[0378] (3) Reactions
[0379] The bacterial systems require, in addition to reduced
flavin, five polypeptides to complete the bioluminescent reaction:
two subunits, a and A, of bacterial luciferin and three units of a
fatty acid reductase system complex, which supplies the
tetradecanal aldehyde. Examples of bacterial bioluminescent systems
useful in the apparatus and methods provided herein include those
derived from Vibrio fisheri and Vibrio harveyi. One advantage to
this system is its ability to operate at cold temperatures; certain
surgical procedures are performed by cooling the body to lower
temperatures.
[0380] Bacterial luciferase catalyzes the flavin-mediated
hydroxylation of a long-chain aldehyde to yield carboxylic acid and
an excited flavin; the flavin decays to ground state with the
concomitant emission of blue green light (.lambda. max=490 nm; see,
e.g., Legocki et al. (1986) Proc. Natl. Acad. Sci. USA 81:9080; see
U.S. Pat. No. 5,196,524): 9
[0381] The reaction can be initiated by contacting reduced flavin
mononucleotide (FMNH.sub.2) with a mixture of the bacterial
luciferase, oxygen, and a long-chain aldehyde, usually n-decyl
aldehyde.
[0382] DNA encoding luciferase from the fluorescent bacterium
Alteromonas hanedai is known (CHISSO CORP; see, also, Japanese
application JP 7222590, published Aug. 22, 1995). The reduced
flavin mononucleotide (FMNH.sub.2; luciferin) reacts with oxygen in
the presence of bacterial luciferase to produce an intermediate
peroxy flavin. This intermediate reacts with a long-chain aldehyde
(tetradecanal) to form the acid and the luciferase-bound hydroxy
flavin in its excited state. The excited luciferase-bound hydroxy
flavin then emits light and dissociates from the luciferase as the
oxidized flavin mononucleotide (FMN) and water. In vivo FMN is
reduced again and recycled, and the aldehyde is regenerated from
the acid.
[0383] Flavin reductases have been cloned (see, e.g., U.S. Pat. No.
5,484,723; see, SEQ ID NO. 14 for a representative sequence from
this patent). These as well as NAD(P)H can be included in the
reaction to regenerate FMNH.sub.2 for reaction with the bacterial
luciferase and long chain aldehyde. The flavin reductase catalyzes
the reaction of FMN, which is the luciferase reaction, into
FMNH.sub.2; thus, if luciferase and the reductase are included in
the reaction system, it is possible to maintain the bioluminescent
reaction. Namely, since the bacterial luciferase turns over many
times, bioluminescence continues as long as a long chain aldehyde
is present in the reaction system.
[0384] The color of light produced by bioluminescent bacteria also
results from the participation of a protein blue-florescent protein
(BFP) in the bioluminescence reaction. This protein, which is well
known (see, e.g., Lee et al. (1978) Methods in Enzymology
LVII:226-234), may also be added to bacterial bioluminescence
reactions in order to cause a shift in the color.
[0385] b. Dinoflagellate Bioluminescence Generating Systems
[0386] In dinoflagellates, bioluminescence occurs in organelles
termed scintillons. These organelles are outpocketings of the
cytoplasm into the cell vacuole. The scintillons contain only
dinoflagellate luciferase and luciferin (with its binding protein),
other cytoplasmic components being somehow excluded. The
dinoflagellate luciferin is a tetrapyrrole related to chlorophyll:
10
[0387] or an analog thereof.
[0388] The luciferase is a 135 kD single chain protein that is
active at pH 6.5, but inactive at pH 8 (see, e.g., Hastings (1981)
Bioluminescence and Chemiluminescence, DeLuca et al., eds. Academic
Press, NY, pp.343-360). Luminescent activity can be obtained in
extracts made at pH 8 by simply shifting the pH from 8 to 6. This
occurs in soluble and particulate fractions. Within the intact
scintillon, the luminescent flash occurs for .about.100 msec, which
is the duration of the flash in vivo. In solution, the kinetics are
dependent on dilution, as in any enzymatic reaction. At pH 8, the
luciferin is bound to a protein (luciferin binding protein) that
prevents reaction of the luciferin with the luciferase. At pH 6,
however, the luciferin is released and free to react with the
enzyme.
[0389] D. Isolation and Identification of Nucleic Acids Encoding
Luciferases and GFPs
[0390] Nucleic acid encoding bioluminescent proteins are provided.
Particularly, nucleic acid encoding Renilla reniformis GFP is
provided.
[0391] 1. Isolation of Specimens of the Genus Renilla
[0392] Specimens of Renilla are readily available from the oceans
of the world, including the Gulf of Mexico, Pacific Ocean and
Atlantic Ocean. Renilla typically live on the ocean bottom at about
30 to 100 feet deep and can be easily collected by dragging. For
example, specimens of R. kollikeri can be obtained off the coast of
California or Baja, Mexico. Alternatively, live specimens of
Renilla may be purchased from a commercial supplier (e.g., Gulf
Marine Incorporated, Panacea, Fla.). Upon capture or receipt, the
specimens are washed thoroughly and may also be dissected to enrich
for light-emitting tissues. The whole organisms or dissected
tissues are then snap frozen and stored in liquid nitrogen.
[0393] As described in detail in the examples below, the frozen
tissues were used as a source to isolate nucleic acids encoding
Renilla mulleri GFP and luciferase (e.g., see SEQ ID NO. 15 and SEQ
ID NO. 17, respectively).
[0394] 2. Preparation of Renilla cDNA Expression Libraries
[0395] Renilla cDNA expression libraries may be prepared from
intact RNA following the methods described herein or by other
methods known to those of skill the art (e.g., see Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; U.S. Pat. No.
5,292,658).
[0396] Typically, the preparation of cDNA libraries includes the
isolation of polyadenylated RNA from the selected organism followed
by single-strand DNA synthesis using reverse transcriptase,
digestion of the RNA strand of the DNA/RNA hybrid and subsequent
conversion of the single-stranded DNA to double stranded cDNA.
[0397] a. RNA Isolation and cDNA Synthesis
[0398] Whole Renilla or dissected Renilla tissues can be used a
source of total cytoplasmic RNA for the preparation of Renilla
cDNA. Total intact RNA can be isolated from crushed Renilla tissue,
for example, by using a modification of methods generally known in
the art (e.g., see Chirgwin et al. (1970) Biochemistry
18:5294-5299). After isolating total cellular RNA, polyadenylated
RNA species are then easily separated from the nonpolyadenylated
species using affinity chromatography on oligodeoxythymidylate
cellulose columns, (e.g., as described by Aviv et al., (1972) Proc.
Natl. Acad. Sci. U.S.A. 69:1408).
[0399] The purified Renilla polyA-mRNA is then subjected to a cDNA
synthesis reaction to generate a cDNA library from total
polyA-mRNA. Briefly, reverse transcriptase is used to extend an
annealed polydT primer to generate an RNA/DNA duplex. The RNA
strand is then digested using an RNase, e.g., RNase H, and
following second-strand synthesis, the cDNA molecules are
blunted-ended with S1 nuclease or other appropriate nuclease. The
resulting double-stranded cDNA fragments can be ligated directly
into a suitable expression vector or, alternatively,
oligonucleotide linkers encoding restriction endonuclease sites can
be ligated to the 5'-ends of the cDNA molecules to facilitate
cloning of the cDNA fragments.
[0400] b. Construction of cDNA Expression Libraries
[0401] The best characterized vectors for the construction of cDNA
expression libraries are lambda vectors. Lambda-based vectors
tolerate cDNA inserts of about 12 kb and provide greater ease in
library screening, amplification and storage compared to standard
plasmid vectors. Presently preferred vectors for the preparation of
Renilla cDNA expression libraries are the Lambda, Uni-Zap,
Lambda-Zap II or Lambda-ZAP Express/EcoRI/XhoI vectors, which are
known to those of skill in the art (e.g., see U.S. Pat. No.
5,128,256), and are also commercially available (Stratagene, La
Jolla, Calif.).
[0402] Generally, the Lambda-Zap vectors combine the high
efficiency of a bacteriophage lambda vector systems with the
versatility of a plasmid system. Fragments cloned into these
vectors can be automatically excised using a helper phage and
recircularized to generate subclones in the pBK-derived phagemid.
The pBK phagemid carries the neomycin-resistance gene for selection
in bacteria and G418 selection in eukaryotic cells or may contain
the .beta.-lactamase resistance gene. Expression of the recombinant
polypeptide is under the control of the lacZ promoter in bacteria
and the CMV promoter in eukaryotes.
[0403] More specifically, these lambda-based vectors are composed
of an initiator-terminator cassette containing the plasmid system,
e.g., a pBK Bluescript derivative (Stratagene, San Diego),
bracketed by the right and left arm of the bacteriophage lambda.
The lambda arms allow for efficient packaging of replicated DNA
whereas the excisable initiator-terminator cassette allows for easy
cloning of the cDNA fragments and the generation of a plasmid
library without the need for additional subcloning.
[0404] When used herein, cDNA fragments are inserted into the
multiple cloning site contained within the initiator-terminator
cassette of the Lambda-Zap vector to create a set of cDNA
expression vectors. The set of cDNA expression vectors is allowed
to infect suitable E. coli cells, followed by co-infection with a
filamentous helper phage. Within the cell, trans-acting proteins
encoded by the helper phage, e.g., the gene II protein of M13,
recognize two separate domains positioned within the lambda arms of
the vector and introduce single-stranded nicks flanking the
intiator-terminator cassette. Upon a subsequent round of DNA
synthesis, a new DNA strand is synthesized that displaces the
existing nick strand liberating the initiator-terminator cassette.
The displaced strand is then circularized, packaged as filamentous
phage by the helper proteins and excreted from the cell. The BK
plasmid containing the cDNA is recovered by infecting an F' strain
of E. coli and plating the infected cells on solid medium
supplemented with kanamycin for the selection of pBK-containing
cells.
[0405] The Renilla cDNA expression library can be screened using a
variety of methods known to those of skill in the art. For example,
identification of Renilla GFP may be achieved using a functional
screening method employing blue light and observing colonies
visually for emission of green fluorescence or by observing light
emission using one or more bandpass filter.
[0406] 3. Cloning of Renilla reniformis Green Fluorescent
Protein
[0407] Renilla reniformis GFP has 233 amino aids compared to GFPs
from animals that contain luciferase-GFP bioluminescent systems
Renilla mulleri, Ptilosarcus and Aequorea victoria. Other such GFPs
have 238 amino acids. At the amino acid level, Renilla reniformis
is respectively 53, 51 and 19% identical to the GFPs from these
animals. The extent of identity of Renilla reniformis GFP to the
half dozen cloned anthozoan coral GFPs, which do not contain
associated luciferases, ranges from 32 to 38%. The overall identity
among these GFPs is surprisingly low for a protein evolved from a
common ancestor. These relationships are depicted as a phylogenetic
tree (FIG. 1).
[0408] Most surprising is the finding that the Renilla reniformis
GFP is much more closely related to Ptilosarcus GFP (77% identity)
than to Renilla reniformis GFP (53%). It is unclear why the
sequence relatedness between these 3 GFPs does not follow
traditional taxonomy. Given the sequence differences at the amino
acid level, coding DNA sequences are surprisingly well conserved.
Renilla reniformis GFP DNA is 56 and 59% identical to Renilla
mulleri and Ptilosarcus GFP DNA.
[0409] Thus cloning Renilla reniformis GFP clone suggests why many
groups may have failed in attempts to clone this gene by
traditional methods. An attempt to sequence the entire protein by
Edman degradation was difficult from the outset because the GFP was
refractory to most attempts at specific proteolysis. Although over
80% of the protein was eventually accurately sequenced, a 30 amino
acid region (110-139 of SEQ ID NO. 27) had not be sequenced (as
well as other regions, including amino acids 41-43, 65-71; SEQ ID
NO. 27). This 30 amino acid region apparently is degraded by the
proteolytic methods used into very small fragments that are
difficult to isolate and sequence; proper ordering of sequenced
fragments was also difficult.
[0410] The cloned DNA fragments can be replicated in bacterial
cells, such as E. coli. A preferred DNA fragment also includes a
bacterial origin of replication, to ensure the maintenance of the
DNA fragment from generation to generation of the bacteria. In this
way, large quantities of the DNA fragment can be produced by
replication in bacteria. Preferred bacterial origins of replication
include, but are not limited to, the f1-ori and col E1 origins of
replication. Preferred hosts contain chromosomal copies of DNA
encoding T7 RNA polymerase operably linked to an inducible
promoter, such as the lacUV promoter (see, U.S. Pat. No.
4,952,496). Such hosts include, but are not limited to, lysogens E.
coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21
(DE3). Strain BL21 (DE3) is preferred. The pLys strains provide low
levels of T7 lysozyme, a natural inhibitor of T7 RNA
polymerase.
[0411] For expression and for preparation of muteins, such as
temperature sensitive muteins, eukaryotic cells, among them, yeast
cells, such as Saccharomyces are preferred.
[0412] Nucleic acid encoding fusion proteins of the luciferases and
GFPs are also provided. The resulting fusion proteins are also
provided. Nucleic acids that encode luciferase and GFPs as
polycistronic mRNA or under the control of separate promoters are
also provided. Methods of use thereof are also provided.
[0413] The GFP cloned from Renilla has spectral properties that
make it extremely useful. These properties include very high
quantum efficiency, high molar absorbency and efficient use with
universally available fluorescein filters (e.g., Endo GFP filter
set sold by Chroma). It is known that Renilla reniformis GFP is
sixfold brighter than the wild-type Aequorea GFP on a molar basis,
and three to fourfold brighter than the brightest mutant.
[0414] The Renilla mullerei GFP encoded by the nucleic acid clones
provided herein exhibits similar functional characteristics, and
the spectra appear identical with those from native reniformis GFP.
Sequence comparison among the GFPs isolated from Aequorea victoria,
Renilla mullerei, and Ptilosarcus reveal that the chromophore
sequences of R. mullerei and Ptilosarcus are identical, and differ
from A. victoria. These sequence differences point to protein sites
that can be modified without affecting the essential fluorescence
properties and also provide a means to identify residues that
change these properties.
[0415] 4. Isolation and Identification of DNA Encoding Renilla
mulleri GFP
[0416] Methods for identification and cloning of GPFs from Renilla
have been described (see, published International PCT application
No. WO 99/49019, and copending allowed U.S. application Ser. No.
09/277,716). Nucleic acid encoding Renilla mulleri has been
isolated. Briefly, a R. mulleri .lambda. Uni-Zap cDNA expression
plasmid library was prepared, transformed into competent E. coli
cells and plated onto modified L-broth plates containing carbon
black to absorb background fluorescence. Transformants were sprayed
with a solution containing IPTG to induce expression of the
recombinant Renilla GFP from the heterologous cDNA. To identify GFP
expressing clones, transformants were placed in blue light,
preferably 470 to 490 nm light, and colonies that emitted green
fluorescence were isolated and grown in pure culture.
[0417] The nucleotide sequence of the cDNA insert of a green
fluorescent transformant was determined (e.g., see SEQ ID NO. 15).
The 1,079 cDNA insert encodes a 238 amino acid polypeptide that is
only 23.5% identical to A. victoria GFP. The recombinant protein
exhibits excitation and emission spectra similar to those reported
for live Renilla species.
[0418] 5. Isolation and Identification of DNA Encoding Renilla
mulleri Luciferase
[0419] The above-described R. mulleri cDNA expression library was
also used to clone DNA encoding a R. mulleri luciferase. Single
colony transformants were grown on modified L-broth plates
containing carbon black and expression from the heterologous DNA
was induced with IPTG, essentially as described above. After
allowing time for expression, the transformants were sprayed with
coelenterazine and screened for those colonies that emit blue
light. Light-emitting colonies were isolated and grown in pure
culture.
[0420] The nucleotide sequence of the cDNA insert contained in the
light-emitting transformant was determined. The 1,217 cDNA insert
encodes a 311 amino acid polypeptide. The recombinant protein
exhibits excitation and emission spectra similar to those reported
for live Renilla species.
[0421] E. Recombinant Expression of Proteins
[0422] 1. DNA Encoding Renilla Proteins
[0423] As described above, DNA encoding a Renilla GFP or Renilla
luciferase can be isolated from natural sources, synthesized based
on Renilla sequences provided herein or isolated as described
herein.
[0424] In preferred embodiments, the DNA fragment encoding a
Renilla GFP has the sequence of amino acids set forth in SEQ ID NO.
27, encoded by nucleic acid, such as that set forth SEQ ID NOs.
23-26 and 27.
[0425] A DNA molecule encoding a Renilla luciferase has the
sequence of amino acids set forth in SEQ ID NO. 18. In more
preferred embodiments, the DNA fragment encodes the sequence of
amino acids encoded by nucleotides 31-963 of the sequence of
nucleotides set forth in SEQ ID NO. 17.
[0426] 2. DNA Constructs for Recombinant Production of Renilla
reniformis GFP and Other Proteins
[0427] DNA is introduced into a plasmid for expression in a desired
host. In preferred embodiments, the host is a bacterial host. The
sequences of nucleotides in the plasmids that are regulatory
regions, such as promoters and operators, are operationally
associated with one another for transcription of the sequence of
nucleotides that encode a Renilla GFP or luciferase. The sequence
of nucleotides encoding the FGF mutein may also include DNA
encoding a secretion signal, whereby the resulting peptide is a
precursor of the Renilla GFP.
[0428] In preferred embodiments the DNA plasmids also include a
transcription terminator sequence. The promoter regions and
transcription terminators are each independently selected from the
same or different genes.
[0429] A wide variety of multipurpose vectors suitable for the
expression of heterologous proteins are known to those of skill in
the art and are commercially available. Expression vectors
containing inducible promoters or constitutive promoters that are
linked to regulatory regions are preferred. Such promoters include,
but are not limited to, the T7 phage promoter and other T7-like
phage promoters, such as the T3, T5 and SP6 promoters, the trp,
Ipp, tet and lac promoters, such as the lacUV5, from E. coli; the
SV40 promoter; the P10 or polyhedron gene promoter of
baculovirus/insect cell expression systems, retroviral
long-terminal repeats and inducible promoters from other eukaryotic
expression systems.
[0430] Particularly preferred vectors for recombinant expression of
Renilla mulleri in prokaryotic organisms are lac- and T7
promoter-based vectors, such as the well known Bluescript vectors,
which are commercially available (Stratagene, La Jolla,
Calif.).
[0431] 3. Host Organisms for Recombinant Production of Renilla
Proteins
[0432] Host organisms include those organisms in which recombinant
production of heterologous proteins have been carried out, such as,
but not limited to, bacteria (for example, E. coli, yeast (for
example, Saccharomyces cerevisiae and Pichia pastoris), fungi,
baculovirus/insect systems, amphibian cells, mammalian cells, plant
cells and insect cells. Presently preferred host organisms are
strains of bacteria or yeast. Most preferred host organisms are
strains of E. coli or Saccharomyces cerevisiae.
[0433] 4. Methods for Recombinant Production of Renilla
Proteins
[0434] The DNA encoding a Renilla GFP or Renilla mulleri luciferase
is introduced into a plasmid in operative linkage to an appropriate
promoter for expression of polypeptides in a selected host
organism. The DNA molecule encoding the Renilla GFP or luciferase
may also include a protein secretion signal that functions in the
selected host to direct the mature polypeptide into the periplasm
or culture medium. The resulting Renilla GFP or luciferase can be
purified by methods routinely used in the art, including methods
described hereinafter in the Examples.
[0435] Methods of transforming suitable host cells, preferably
bacterial cells, and more preferably E. coli cells, as well as
methods applicable for culturing said cells containing a gene
encoding a heterologous protein, are generally known in the art.
See, for example, Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0436] Once the Renilla-encoding DNA molecule has been introduced
into the host cell, the desired Renilla GFP is produced by
subjecting the host cell to conditions under which the promoter is
induced, whereby the operatively linked DNA is transcribed. The
cellular extracts of lysed cells containing the protein may be
prepared and the resulting "clarified lysate" was employed as a
source of recombinant Renilla GFP or Renilla mulleri luciferase.
Alternatively, the lysate may be subjected to additional
purification steps (e.g., ion exchange chromatography or
immunoaffinity chromatography) to further enrich the lysate or
provide a homogeneous source of the purified enzyme (see e.g., U.S.
Pat. Nos. 5,292,658 and 5,418,155).
[0437] 5. Recombinant Cells Expressing Heterologous Nucleic Acid
Encoding Renilla GFP
[0438] Cells, vectors and methods are described with respect to
Renilla. The same cells, vectors and methods may be used for
expressing luciferases and other GFPs from species including
Gaussia, Pleuromamma and Ptilosarcus.
[0439] Recombinant cells containing heterologous nucleic acid
encoding a Renilla reniformis GFP are provided. In preferred
embodiments, the recombinant cells express the encoded Renilla GFP
which is functional and non-toxic to the cell.
[0440] In certain embodiments, the recombinant cells may also
include heterologous nucleic acid encoding a component of a
bioluminescence-generating system, preferably a photoprotein or
luciferase. In preferred embodiments, the nucleic acid encoding the
bioluminescence-generating system component is isolated from the
species Aequorea, Vargula or Renilla. In more preferred
embodiments, the bioluminescence-generating system component is a
Renilla mulleri luciferase having the amino acid sequence set forth
in SEQ ID NO. 18.
[0441] Recombinant host cells containing heterologous nucleic acid
encoding a Renilla mulleri luciferase are also provided. In
preferred embodiments, the heterologous nucleic acid encodes the
sequence of amino acids as set forth in SEQ ID NO. 18. In more
preferred embodiments, the heterologous nucleic acid encodes the
sequence of nucleotides set forth in SEQ ID NO. 17.
[0442] Exemplary cells include bacteria (e.g., E. coli), plant
cells, cells of mammalian origin (e.g., COS cells, mouse L cells,
Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK)
cells, African green monkey cells and other such cells known to
those of skill in the art), amphibian cells (e.g., Xenopus laevis
oocytes), yeast cells (e.g., Saccharomyces cerevisiae, Pichia
pastoris), and the like. Exemplary cells for expressing injected
RNA transcripts include Xenopus laevis oocytes. Eukaryotic cells
that are preferred for transfection of DNA are known to those of
skill in the art or may be empirically identified, and include
HEK293 (which are available from ATCC under accession #CRL 1573);
Ltk.sup.- cells (which are available from ATCC under accession
#CCL1.3); COS-7 cells (which are available from ATCC under
accession #CRL 1651); and DG44 cells (dhfr.sup.- CHO cells; see,
e.g., Urlaub et al. (1986) Cell. Molec. Genet. 12: 555). Presently
preferred cells include strains of bacteria and yeast.
[0443] The recombinant cells that contain the heterologous DNA
encoding the Renilla GFP are produced by transfection with DNA
encoding a Renilla GFP or luciferase or by introduction of RNA
transcripts of DNA encoding a Renilla proteins using methods well
known to those of skill in the art. The DNA may be introduced as a
linear DNA fragment or may be included in an expression vector for
stable or transient expression of the encoding DNA. The sequences
set forth herein for Renilla reniformis GFP are presently preferred
(see SEQ ID NOs 23-25 and 27; see, also SEQ ID NO. 26, which sets
forth human optimized codons).
[0444] Heterologous DNA may be maintained in the cell as an
episomal element or may be integrated into chromosomal DNA of the
cell. The resulting recombinant cells may then be cultured or
subcultured (or passaged, in the case of mammalian cells) from such
a culture or a subculture thereof. Also, DNA may be stably
incorporated into cells or may be transiently expressed using
methods known in the art.
[0445] The recombinant cells can be used in a wide variety of
cell-based assay methods, such as those methods described for cells
expressing wild type or modified A. victoria GFPs or GFP fusion
proteins (e.g., see U.S. Pat. No. 5,625,048; International patent
application Publication Nos. WO 95/21191; WO 96/23810; WO 96/27675;
WO 97/26333; WO 97/28261; WO 97/41228; and WO 98/02571).
[0446] F. Compositions and Conjugates
[0447] Compositions and conjugates and methods of use are described
with reference to Renilla proteins and nucleic acids. The same
compositions and methods for preparation and use thereof are
intended for use with other luciferases, such as Pleuromamma and
Ptilosarcus proteins and nucleic acids.
[0448] 1. Renilla GFP Compositions
[0449] Compositions containing a Renilla GFP or GFP peptide are
provided. The compositions can take any of a number of forms,
depending on the intended method of use therefor. In certain
embodiments, for example, the compositions contain a Renilla GFP or
GFP peptide, preferably Renilla mulleri GFP or Renilla reniformis
GFP peptide, formulated for use in luminescent novelty items,
immunoassays, FRET and FET assays. The compositions may also be
used in conjunction with multi-well assay devices containing
integrated photodetectors, such as those described herein.
[0450] Compositions that contain a Renilla mulleri GFP or GFP
peptide and at least one component of a bioluminescence-generating
system, preferably a luciferase, luciferin or a luciferase and a
luciferin, are provided. In preferred embodiments, the
luciferase/luciferin bioluminescence-generatin- g system is
selected from those isolated from: an insect system, a coelenterate
system, a ctenophore system, a bacterial system, a mollusk system,
a crustacea system, a fish system, an annelid system, and an
earthworm system. Presently preferred bioluminescence-generating
systems are those isolated from Renilla, Aequorea, and Vargula.
[0451] In more preferred embodiments, the
bioluminescence-generating system component is a Renilla mulleri
luciferase having the amino acid sequence set forth in SEQ ID NO.
18 or a Renilla reniformis luciferase. These compositions can be
used in a variety of methods and systems, such as included in
conjunction with diagnostic systems for the in vivo detection of
neoplastic tissues and other tissues, such as those methods
described in detail below.
[0452] These methods and products include any known to those of
skill in the art in which luciferase is used, including, but not
limited to U.S. application Ser. Nos. 08/757,046, 08/597,274 and
08/990,103, U.S. Pat. No. 5,625,048; International patent
application Publication Nos. WO 95/21191; WO 96/23810; WO 96/27675;
WO 97/26333; WO 97/28261; WO 97/41228; and WO 98/02571).
[0453] 2. Renilla Luciferase Compositions
[0454] DNA encoding the Renilla mulleri luciferase or Renilla
reniformis luciferase is used to produce the encoded luciferase,
which has diagnostic applications as well as use as a component of
the bioluminescence generating systems as described herein, such as
in beverages, and methods of diagnosis of neoplasia and in the
diagnostic chips described herein. These methods and products
include any known to those of skill in the art in which luciferase
is used, including, but not limited to, U.S. application Ser. Nos.
08/757,046, 08/597,274 and 08/990,103, U.S. Pat. No. 5,625,048;
International patent application Publication Nos. WO 95/21191; WO
96/23810; WO 96/27675; WO 97/26333; WO 97/28261; WO 97/41228; and
WO 98/02571).
[0455] In other embodiments, the Renilla luciferase and the
remaining components may be packaged as separate compositions,
that, upon mixing, glow. For example, a composition containing
Renilla luciferase may be provided separately from, and use with, a
separate composition containing a bioluminescence substrate and
bioluminescence activator. In another instance, luciferase and
luciferin compositions may be separately provided and the
bioluminescence activator may be added after, or simultaneously
with, mixing of the other two compositions.
[0456] 3. Conjugates
[0457] Conjugates are provided herein for a variety of uses. Among
them are for targeting to tumors for visualization of the tumors,
particularly in situ during surgery. A general description of these
conjugates and the uses thereof is described in allowed U.S.
application Ser. No. 08/908,909. In practice, prior to a surgical
procedure, the conjugate is administered via any suitable route,
whereby the targeting agent binds to the targeted tissue by virtue
of its specific interaction with a tissue-specific cell surface
protein. During surgery the tissue is contacted, with the remaining
component(s), typically by spraying the area or local injection,
and any tissue to which conjugate is bound will glow. The glow
should be sufficient to see under dim light or, if necessary, in
the dark.
[0458] The conjugates that are provided herein contain a targeting
agent, such as a tissue specific or tumor specific monoclonal
antibody or fragment thereof linked either directly or via a linker
to a targeted agent, a Renilla GFP, Renilla or Gaussia luciferase
and other luciferases (including photoproteins or luciferase
enzymes) or a luciferin. The targeted agent may be coupled to a
microcarrier. The linking is effected either chemically, by
recombinant expression of a fusion protein in instances when the
targeted agent is a protein, and by combinations of chemical and
recombinant expression. The targeting agent is one that will
preferentially bind to a selected tissue or cell type, such as a
tumor cell surface antigen or other tissue specific antigen.
[0459] Methods for preparing conjugates are known to those of skill
in the art. For example, aequorin that is designed for conjugation
and conjugates containing such aequorin have been produced (see,
e.g., International PCT application No. WO 94/18342; see, also
Smith et al. (1995) in American Biotechnology Laboratory). Aequorin
has been conjugated to an antibody molecule by means of a
sulfhydryl-reacting binding agent (Stultz et al. (1992) Use of
Recombinant Biotinylated Apoaequorin from Escherichia coli.
Biochemistry 31, 1433-1442). Such methods may be adapted for use
herein to produce the luciferase coupled to protein or other such
molecules, which are useful as targeting agents. Vargula luciferase
has also been linked to other molecules (see, e.g., Japanese
application No. JP 5064583, Mar. 19, 1993). Such methods may be
adapted for use herein to produce luciferase coupled to molecules
that are useful as targeting agents.
[0460] The conjugates can be employed to detect the presence of or
quantitate a particular antigen in a biological sample by direct
correlation to the light emitted from the bioluminescent
reaction.
[0461] As an alternative, a component of the bioluminescence
generating system may be modified for linkage, such as by addition
of amino acid residues that are particularly suitable for linkage
to the selected substrate. This can be readily effected by
modifying the DNA and expressing such modified DNA to produce
luciferase with additional residues at the N- or C-terminus.
[0462] Methods for preparing conjugates are known to those of skill
in the art. For example, aequorin that is designed for conjugation
and conjugates containing such aequorin have been produced (see,
e.g., International PCT application No. WO 94/18342; see, also
Smith et al. (1995) in American Biotechnology Laboratory). Aequorin
has been conjugated to an antibody molecule by means of a
sulfhydryl-reacting binding agent (Stultz et al. (1992) Use of
Recombinant Biotinylated Apoaequorin from Escherichia coli.
Biochemistry 31, 1433-1442). Such methods may be adapted for use
herein to produce aequorin coupled to protein or other such
molecules, which are useful as targeting agents. Vargula luciferase
has also been linked to other molecules (see, e.g., Japanese
application No. JP 5064583, Mar. 19, 1993). Such methods may be
adapted for use herein to produce aequorin coupled to protein or
other such molecules, which are useful as targeting agents. The
bioluminescence generating reactions are used with the Renilla
reniformis GFP provided herein.
[0463] a. Linkers
[0464] Any linker known to those of skill in the art may be used
herein. Other linkers are suitable for incorporation into
chemically produced conjugates. Linkers that are suitable for
chemically linked conjugates include disulfide bonds, thioether
bonds, hindered disulfide bonds, and covalent bonds between free
reactive groups, such as amine and thiol groups. These bonds are
produced using heterobifunctional reagents to produce reactive
thiol groups on one or both of the polypeptides and then reacting
the thiol groups on one polypeptide with reactive thiol groups or
amine groups to which reactive maleimido groups or thiol groups can
be attached on the other. Other linkers include, acid cleavable
linkers, such as bismaleimideothoxy propane, acid
labile-transferrin conjugates and adipic acid diihydrazide, that
would be cleaved in more acidic intracellular compartments; cross
linkers that are cleaved upon exposure to UV or visible light and
linkers, such as the various domains, such as C.sub.H1, C.sub.H2,
and C.sub.H3, from the constant region of human IgG.sub.1 (see,
Batra et al. (1993) Molecular Immunol. 30:379-386). In some
embodiments, several linkers may be included in order to take
advantage of desired properties of each linker.
[0465] Chemical linkers and peptide linkers may be inserted by
covalently coupling the linker to the TA and the targeted agent.
The heterobifunctional agents, described below, may be used to
effect such covalent coupling. Peptide linkers may also be linked
by expressing DNA encoding the linker and TA, linker and targeted
agent, or linker, targeted agent and TA as a fusion protein.
[0466] Flexible linkers and linkers that increase solubility of the
conjugates are contemplated for use, either alone or with other
linkers are contemplated herein.
[0467] Numerous heterobifunctional cross-linking reagents that are
used to form covalent bonds between amino groups and thiol groups
and to introduce thiol groups into proteins, are known to those of
skill in this art (see, e.g., the PIERCE CATALOG, ImmunoTechnology
Catalog & Handbook, 1992-1993, which describes the preparation
of and use of such reagents and provides a commercial source for
such reagents; see, also, e.g., Cumber et al. (1992) Bioconjugate
Chem. 3:397-401; Thorpe et al. (1987) Cancer Res. 47:5924-5931;
Gordon et al. (1987) Proc. Natl. Acad. Sci. 84:308-312; Walden et
al. (1986) J. Mol. Cell Immunol. 2:191-197; Carlsson et al. (1978)
Biochem. J. 173: 723-737; Mahan et al. (1987) Anal. Biochem.
162:163-170; Wawryznaczak et al. (1992) Br. J. Cancer 66:361-366;
Fattom et al. (1992) Infection & Immun. 60:584-589). These
reagents may be used to form covalent bonds between the TA and
targeted agent. These reagents include, but are not limited to:
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; disulfide
linker); sulfosuccinimidyl
6-(3-(2-pyridyidithio)propionamido)hexanoate (sulfo-LC-SPDP);
succinimidyloxycarbonyl-.alpha.-methyl benzyl thiosulfate (SMBT,
hindered disulfate linker); succinimidyl
6-(3-(2-pyridyidithio)propionamido)hexanoate (LC-SPDP);
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sulfo-SMCC); succinimidyl 3-(2-pyridyidithio)-butyrate (SPDB;
hindered disulfide bond linker); sulfosuccinimidyl
2-(7-azido-4-methylcoumarin-3-a-cetamide)ethyl-1,3'-dit-
hiopropionate (SAED); sulfo-succinimidyl
7-azido-4-methylcoumarin-3-acetat- e (SAMCA); sulfosuccinimidyl
6-(alpha-methyl-alpha-(2-pyridyldithio)toluam- ido)-hexanoate
(sulfo-LC-SMPT); 1,4-di-(3'-(2'-pyridyidithio)propion-amido-
)butane (DPDPB);
4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyrid-
ylthio)-toluene (SMPT, hindered disulfate
linker);sulfosuccinimidyl6(.alph-
a.-methy-1-.alpha.-(2-pyridyldithio)toluamido)hexanoate
(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS); m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester
(sulfo-MBS); N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB;
thioether linker); sulfosuccinimidyl(4-iodoacetyl)amino benzoate
(sulfo-SIAB); succinimidyl4(p-maleimidophenyl)butyrate (SMPB);
sulfosuccinimidyl4-(p-ma- -leimidophenyl)butyrate (sulfo-SMPB);
azidobenzoyl hydrazide (ABH).
[0468] Acid cleavable linkers, photocleavable and heat sensitive
linkers may also be used, particularly where it may be necessary to
cleave the targeted agent to permit it to be more readily
accessible to reaction. Acid cleavable linkers include, but are not
limited to, bismaleimideothoxy propane; and adipic acid dihydrazide
linkers (see, e.g., Fattom et al. (1992) Infection & Immun.
60:584-589) and acid labile transferrin conjugates that contain a
sufficient portion of transferrin to permit entry into the
intracellular transferrin cycling pathway (see, e.g., Welhoner et
al. (1991) J. Biol. Chem. 266:4309-4314).
[0469] Photocleavable linkers are linkers that are cleaved upon
exposure to light (see, e.g., Goldmacher et al. (1992) Bioconj.
Chem. 3:104-107, which linkers are herein incorporated by
reference), thereby releasing the targeted agent upon exposure to
light. Photocleavable linkers that are cleaved upon exposure to
light are known (see, e.g., Hazum et al. (1981) in Pept. Proc. Eur.
Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, which describes
the use of a nitrobenzyl group as a photocleavable protective group
for cysteine; Yen et al. (1989) Makromol. Chem 190:69-82, which
describes water soluble photocleavable copolymers, including
hydroxypropylmethacrylamide copolymer, glycine copolymer,
fluorescein copolymer and methylrhodamine copolymer; Goldmacher et
al. (1992) Bioconj. Chem. 3:104-107, which describes a cross-linker
and reagent that undergoes photolytic degradation upon exposure to
near UV light (350 nm); and Senter et al. (1985) Photochem.
Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl
chloride cross linking reagents that produce photocleavable
linkages), thereby releasing the targeted agent upon exposure to
light. Such linkers would have particular use in treating
dermatological or ophthalmic conditions that can be exposed to
light using fiber optics. After administration of the conjugate,
the eye or skin or other body part can be exposed to light,
resulting in release of the targeted moiety from the conjugate.
Such photocleavable linkers are useful in connection with
diagnostic protocols in which it is desirable to remove the
targeting agent to permit rapid clearance from the body of the
animal.
[0470] b. Targeting Agents
[0471] Targeting agents include any agent that will interact with
and localize the targeted agent cells in a tumor or specialized
tissue (targeted tissue). Such agents include any agent that
specifically interacts with a cell surface protein or receptor that
is present at sufficiently higher concentrations or amounts on the
targeted tissue, whereby, when contacted with an appropriate
bioluminescence generating reagent and activators produces light.
These agents include, but are not limited to, growth factors,
preferentially modified to not internalize, methotrexate, and
antibodies, particularly, antibodies raised against tumor specific
antigens. A plethora of tumor-specific antigens have been
identified from a number of human neoplasms.
[0472] c. Anti-Tumor Antigen Antibodies
[0473] Polyclonal and monoclonal antibodies produced against
selected antigens. Alternatively, many such antibodies are
presently available. An exemplary list of antibodies and the tumor
antigen for which each has been directed against is provided in
U.S. application Ser. No. 08/908,909, which is incorporated by
reference in its entirety. It is contemplated that any of the
antibodies listed may be conjugated with a bioluminescence
generating component following the methods provided herein.
[0474] Among the preferred antibodies for use in the methods herein
are those of human origin or, more preferably, are humanized
monoclonal antibodies. These are preferred for diagnosis of
humans.
[0475] d. Preparation of the Conjugates
[0476] The methods for preparation of the conjugates for use in the
tumor diagnostic methods can be used for preparation of the fusion
proteins and conjugated proteins for use in the BRET system
desribed below. Any method for linking proteins may be used. For
example, methods for linking a luciferase to an antibody is
described in U.S. Pat. No. 5,486,455. As noted above, the targeting
agent and luciferin or luciferase may be linked directly, such as
through covalent bonds, i.e., sulfhyryl bonds or other suitable
bonds, or they may be linked through a linker. There may be more
than one luciferase or luciferin per targeting agent, or more than
one targeting agent per luciferase or luciferin.
[0477] Alternatively, an antibody, or F(Ab).sub.2 antigen-binding
fragment thereof or other protein targeting agent may be fused
(directly or via a linking peptide) to the luciferase using
recombinant DNA technology. For example, the DNA encoding any of
the anti-tumor antibodies of Table 3 may be ligated in the same
translational reading frame to DNA encoding any of the
above-described luciferases, e.g., SEQ ID NOs. 1-14 and inserted
into an expression vector. The DNA encoding the recombinant
antibody-luciferase fusion may be introduced into an appropriate
host, such as bacteria or yeast, for expression.
[0478] 4. Formulation of the Compositions for Use in the Diagnostic
Systems
[0479] In most embodiments, the Renilla GFPS and components of the
diagnostic systems provided herein, such as Renilla luciferase, are
formulated into two compositions: a first composition containing
the conjugate; and a second composition containing the remaining
components of the bioluminescence generating system. The
compositions are formulated in any manner suitable for
administration to an animal, particularly a mammal, and more
particularly a human. Such formulations include those suitable for
topical, local, enteric, parenteral, intracystal, intracutaneous,
intravitreal, subcutaneous, intramuscular, or intravenous
administration.
[0480] For example, the conjugates, which in preferred embodiments,
are a targeting agent linked to a luciferase (or photoprotein) are
formulated for systemic or local administration. The remaining
components are formulated in a separate second composition for
topical or local application. The second composition will typically
contain any other agents, such as spectral shifters that will be
included in the reaction. It is preferred that the components of
the second composition are formulated in a time release manner or
in some other manner that prevents degradation and/or interaction
with blood components.
[0481] a. The First Composition: Formulation of the Conjugates
[0482] As noted above, the conjugates either contain a luciferase
or luciferin and a targeting agents. The preferred conjugates are
formed between a targeting agent and a luciferase, particularly the
Gaussia, Renilla mulleri or Pleuromamma luciferase. The conjugates
may be formulated into pharmaceutical compositions suitable for
topical, local, intravenous and systemic application. Effective
concentrations of one or more of the conjugates are mixed with a
suitable pharmaceutical carrier or vehicle. The concentrations or
amounts of the conjugates that are effective requires delivery of
an amount, upon administration, that results in a sufficient amount
of targeted moiety linked to the targeted cells or tissue whereby
the cells or tissue can be visualized during the surgical
procedure. Typically, the compositions are formulated for single
dosage administration. Effective concentrations and amounts may be
determined empirically by testing the conjugates in known in vitro
and in vivo systems, such as those described here; dosages for
humans or other animals may then be extrapolated therefrom.
[0483] Upon mixing or addition of the conjugate(s) with the
vehicle, the resulting mixture may be a solution, suspension,
emulsion or the like. The form of the resulting mixture depends
upon a number of factors, including the intended mode of
administration and the solubility of the conjugate in the selected
carrier or vehicle. The effective concentration is sufficient for
targeting a sufficient amount of targeted agent to the site of
interest, whereby when combined with the remaining reagents during
a surgical procedure the site will glow. Such concentration or
amount may be determined based upon in vitro and/or in vivo data,
such as the data from the mouse xenograft model for tumors or
rabbit ophthalmic model. If necessary, pharmaceutically acceptable
salts or other derivatives of the conjugates may be prepared.
[0484] Pharmaceutical carriers or vehicles suitable for
administration of the conjugates provided herein include any such
carriers known to those skilled in the art to be suitable for the
particular mode of administration. In addition, the conjugates may
be formulated as the sole pharmaceutically ingredient in the
composition or may be combined with other active ingredients.
[0485] The conjugates can be administered by any appropriate route,
for example, orally, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid, semi-liquid or solid form
and are formulated in a manner suitable for each route of
administration. Intravenous or local administration is presently
preferred. Tumors and vascular proliferative disorders, will
typically be visualized by systemic, intradermal or intramuscular,
modes of administration.
[0486] The conjugate is included in the pharmaceutically acceptable
carrier in an amount sufficient to produce detectable tissue and to
not result in undesirable side effects on the patient or animal. It
is understood that number and degree of side effects depends upon
the condition for which the conjugates are administered. For
example, certain toxic and undesirable side effects are tolerated
when trying to diagnose life-threatening illnesses, such as tumors,
that would not be tolerated when diagnosing disorders of lesser
consequence.
[0487] The concentration of conjugate in the composition will
depend on absorption, inactivation and excretion rates thereof, the
dosage schedule, and amount administered as well as other factors
known to those of skill in the art. Typically an effective dosage
should produce a serum concentration of active ingredient of from
about 0.1 ng/ml to about 50-1000 .mu.g/ml, preferably 50-100
.mu.g/ml. The pharmaceutical compositions typically should provide
a dosage of from about 0.01 mg to about 100-2000 mg of conjugate,
depending upon the conjugate selected, per kilogram of body weight
per day. Typically, for intravenous administration a dosage of
about between 0.05 and 1 mg/kg should be sufficient. Local
application for, such as visualization of ophthalmic tissues or
local injection into joints, should provide about 1 ng up to 1000
.mu.g, preferably about 1 .mu.g to about 100 .mu.g, per single
dosage administration. It is understood that the amount to
administer will be a function of the conjugate selected, the
indication, and possibly the side effects that will be tolerated.
Dosages can be empirically determined using recognized models.
[0488] The active ingredient may be administered at once, or may be
divided into a number of smaller doses to be administered at
intervals of time. It is understood that the precise dosage and
duration of administration is a function of the disease condition
being diagnosed and may be determined empirically using known
testing protocols or by extrapolation from in vivo or in vitro test
data. It is to be noted that concentrations and dosage values may
also vary with the severity of the condition to be alleviated. It
is to be further understood that for any particular subject,
specific dosage regimens should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed compositions.
[0489] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include any of the
following components: a sterile diluent, such as water for
injection, saline solution, fixed oil, polyethylene glycol,
glycerine, propylene glycol or other synthetic solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; chelating
agents, such as ethylenediaminetetraacetic acid (EDTA); buffers,
such as acetates, citrates and phosphates; and agents for the
adjustment of tonicity such as sodium chloride or dextrose.
Parental preparations can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass, plastic or other
suitable material.
[0490] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof. Liposomal suspensions may also be suitable as
pharmaceutically acceptable carriers. These may be prepared
according to methods known to those skilled in the art.
[0491] The conjugates may be prepared with carriers that protect
them against rapid elimination from the body, such as time release
formulations or coatings. Such carriers include controlled release
formulations, such as, but not limited to, implants and
microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylacetic
acid and others.
[0492] The conjugates may be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Such solutions, particularly those
intended for ophthalmic use, may be formulated as 0.01%-10%
isotonic solutions, pH about 5-7, with appropriate salts. The
ophthalmic compositions may also include additional components,
such as hyaluronic acid. The conjugates may be formulated as
aerosols for topical application (see, e.g., U.S. Pat. Nos.
4,044,126, 4,414,209, and 4,364,923).
[0493] Also, the compositions for activation of the conjugate in
vivo during surgical procedures may be formulated as an aerosol.
These compositions contain the activators and also the remaining
bioluminescence generating agent, such as luciferin, where the
conjugate targets a luciferase, or a luciferase, where the
conjugate targets a luciferin, such as coelenterazine.
[0494] If oral administration is desired, the conjugate should be
provided in a composition that protects it from the acidic
environment of the stomach. For example, the composition can be
formulated in an enteric coating that maintains its integrity in
the stomach and releases the active compound in the intestine. Oral
compositions will generally include an inert diluent or an edible
carrier and may be compressed into tablets or enclosed in gelatin
capsules. For the purpose of oral administration, the active
compound or compounds can be incorporated with excipients and used
in the form of tablets, capsules or troches. Pharmaceutically
compatible binding agents and adjuvant materials can be included as
part of the composition.
[0495] Tablets, pills, capsules, troches and the like can contain
any of the following ingredients, or compounds of a similar nature:
a binder, such as microcrystalline cellulose, gum tragacanth and
gelatin; an excipient such as starch and lactose, a disintegrating
agent such as, but not limited to, alginic acid and corn starch; a
lubricant such as, but not limited to, magnesium stearate; a
glidant, such as, but not limited to, colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin; and a flavoring
agent such as peppermint, methyl salicylate, and fruit
flavoring.
[0496] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as a
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The conjugates
can also be administered as a component of an elixir, suspension,
syrup, wafer, chewing gum or the like. A syrup may contain, in
addition to the active compounds, sucrose as a sweetening agent and
certain preservatives, dyes and colorings and flavors.
[0497] The active materials can also be mixed with other active
materials that do not impair the desired action, or with materials
that supplement the desired action, such as cis-platin for
treatment of tumors.
[0498] Finally, the compounds may be packaged as articles of
manufacture containing packaging material, one or more conjugates
or compositions as provided herein within the packaging material,
and a label that indicates the indication for which the conjugate
is provided.
[0499] b. The Second Composition
[0500] The second composition will include the remaining components
of the bioluminescence generating reaction. In preferred
embodiments in which these components are administered
systemically, the remaining components include the luciferin or
substrate, and optionally additional agents, such as spectral
shifters, particularly the GFPs provided herein. These components,
such as the luciferin, can be formulated as described above for the
conjugates. In some embodiments, the luciferin or luciferase in
this composition will be linked to a protein carrier or other
carrier to prevent degradation or dissolution into blood cells or
other cellular components.
[0501] For embodiments, in which the second composition is applied
locally or topically, they can be formulated in a spray or aerosol
or other suitable means for local or topical application.
[0502] In certain embodiments described herein, all components,
except an activator are formulated together, such as by
encapsulation in a time release formulation that is targeted to the
tissue. Upon release the composition will have been localized to
the desired site, and will begin to glow.
[0503] In practice, the two compositions can be administered
simultaneously or sequentially. Typically, the first composition,
which contains the conjugate is administered first, generally an
hour or two before the surgery, and the second composition is then
administered, either pre-operatively or during surgery.
[0504] The conjugates that are provided herein contain a targeting
agent, such as a tissue specific or tumor specific monoclonal
antibody or fragment thereof linked either directly or via a linker
to a targeted agent, a luciferase (including photoproteins or
luciferase enzymes) or a luciferin. The targeted agent may be
coupled to a microcarrier. The linking is effected either
chemically, by recombinant expression of a fusion protein in
instances when the targeted agent is a protein, and by combinations
of chemical and recombinant expression. The targeting agent is one
that will preferentially bind to a selected tissue or cell type,
such as a tumor cell surface antigen or other tissue specific
antigen.
[0505] Methods for preparing conjugates are known to those of skill
in the art. For example, aequorin that is designed for conjugation
and conjugates containing such aequorin have been produced (see,
e.g., International PCT application No. WO 94/18342; see, also
Smith et al. (1995) in American Biotechnology Laboratory). Aequorin
has been conjugated to an antibody molecule by means of a
sulfhydryl-reacting binding agent (Stultz et al. (1992) Use of
Recombinant Biotinylated Apoaequorin from Escherichia coli
(Biochemistry 31:1433-1442). Such methods may be adapted for use
herein to produce aequorin coupled to protein or other such
molecules, which are useful as targeting agents. Vargula luciferase
has also been linked to other molecules (see, e.g., Japanese
application No. JP 5064583, Mar. 19, 1993). Such methods may be
adapted for use herein to produce aequorin coupled to protein or
other such molecules, which are useful as targeting agents.
[0506] Aequorin=L -antibody conjugates have been employed to detect
the presence of or quantitate a particular antigen in a biological
sample by direct correlation to the light emitted from the
bioluminescent reaction.
[0507] As an alternative, the Renilla GFP or Renilla mulleri or
Gaussia luciferase or a component of the bioluminescence generating
system may be modified for linkage, such as by addition of amino
acid residues that are particularly suitable for linkage to the
selected substrate. This can be readily effected by modifying the
DNA and expressing such modified DNA to produce luciferase with
additional residues at the N- or C-terminus.
[0508] Selection of the system depends upon factors such as the
desired color and duration of the bioluminescence desired as well
as the particular item. Selection of the targeting agent primarily
depends upon the type and characteristics of neoplasia or tissue to
be visualized and the setting in which visualization will be
performed.
[0509] The Renilla reniformis GFP is added to one or both
compositions to act as a spectral shifter.
[0510] c. Practice of the Reactions in Combination with Targeting
Agents
[0511] The particular manner in which each bioluminescence system
will be combined with a selected targeting agent will be a function
of the agent and the neoplasia or tissue to be visualized. In
general, however, a luciferin, Renilla GFP, Renilla mulleri,
Pleuromamma or Gaussia luciferase or other luciferase, of the
reaction will be conjugated to the targeting agent, administered to
an animal prior to surgery. During the surgery, the tissues of
interest are contacted with the remaining component(s) of a
bioluminescence generating system. Any tissue to which or with
which the targeting agent reacts will glow.
[0512] Any color of visible light produced by a bioluminescence
generating system is contemplated for use in the methods herein.
Preferably the visible light is a combination of blue, green and/or
red light of varying intensities and wavelengths. For visualizing
neoplasia or specialty tissues through mammalian tissues or tumors
deeply embedded in tissue, longer wavelengths of visible light,
ie., red and near infrared light, is preferred because wavelengths
of near infrared light of about 700-1300 nm are known to penetrate
soft tissue and bone (e.g., see U.S. Pat. No. 4,281,645).
[0513] In other embodiments, the conjugate can be applied to the
tissues during surgery, such as by spraying a sterile solution over
the tissues, followed by application of the remaining components.
Tissues that express the targeted antigen will glow.
[0514] The reagents may be provided in compositions, such as
suspensions, as powders, as pastes or any in other suitable sterile
form. They may be provided as sprays, aerosols, or in any suitable
form. The reagents may be linked to a matrix, particularly
microbeads suitable for in vivo use and of size that they pass
through capillaries. Typically all but one or more, though
preferably all but one, of the components necessary for the
reaction will be mixed and provided together; reaction will be
triggered contacting the mixed component(s) with the remaining
component(s), such as by adding Ca.sup.2+, FMN with reductase,
FMNH.sub.2, ATP, air or oxygen.
[0515] In preferred embodiments the luciferase or
luciferase/luciferin will be provided in combination with the
targeting agent before administration to the patient. The targeting
agent conjugate will then be contacted in vivo with the remaining
components. As will become apparent herein, there are a multitude
of ways in which each system may be combined with a selected
targeting agent.
[0516] G. Combinations
[0517] Renilla reniformis GFP can be used in combination with
articles of manufacture to produce novelty items. The Renilla
reniformis GFP can be used with a bioluminescence generating
system. Such items and methods for preparation are described in
detail in U.S. Pat. Nos. 5,876,995, 6,152,358 and 6,113,886) These
novelty items, which are articles of manufacture, are designed for
entertainment, recreation and amusement, and include, but are not
limited to: toys, particularly squirt guns, toy cigarettes, toy
"Halloween" eggs, footbags and board/card games; finger paints and
other paints, slimy play material; textiles, particularly clothing,
such as shirts, hats and sports gear suits, threads and yarns;
bubbles in bubble making toys and other toys that produce bubbles;
balloons; figurines; personal items, such as bath powders, body
lotions, gels, powders and creams, nail polishes, cosmetics
including make-up, toothpastes and other dentifrices, soaps, body
paints, and bubble bath; items such as fishing lures, inks, paper;
foods, such as gelatins, icings and frostings; fish food containing
luciferins and transgenic fish, particularly transgenic fish that
express a luciferase; plant food containing a luciferin or
luciferase, preferably a luciferin for use with transgenic plants
that express luciferase; and beverages, such as beer, wine,
champagne, soft drinks, and ice cubes and ice in other
configurations; fountains, including liquid "fireworks" and other
such jets or sprays or aerosols of compositions that are solutions,
mixtures, suspensions, powders, pastes, particles or other suitable
form.
[0518] Any article of manufacture that can be combined with a
bioluminescence-generating system as provided herein and thereby
provide entertainment, recreation and/or amusement, including use
of the items for recreation or to attract attention, such as for
advertising goods and/or services that are associated with a logo
or trademark is contemplated herein. Such uses may be in addition
to or in conjunction with or in place of the ordinary or normal use
of such items. As a result of the combination, the items glow or
produce, such as in the case of squirt guns and fountains, a
glowing fluid or spray of liquid or particles.
[0519] H. Exemplary Uses of Renilla reniformis GFPs and Encoding
Nucleic Acid Molecules
[0520] 1. Methods for Diagnosis of Neoplasms and Other Tissues
[0521] Methods for diagnosis and visualization of tissues in vivo
or in situ, preferably neoplastic tissue, using compositions
containing a Renilla mulleri or Ptilosarcus GFP and/or a Renilla
mulleri, Pleuromamma or Gaussia luciferase are provided. For
example, the Renilla mulleri GFP protein can be used in conjunction
with diagnostic systems that rely on bioluminescence for
visualizing tissues in situ, such as those described in co-pending
application Ser. No. 08/908,909. The systems are particularly
useful for visualizing and detecting neoplastic tissue and
specialty tissue, such as during non-invasive and invasive
procedures. The systems include compositions containing conjugates
that include a tissue specific, particularly a tumor-specific,
targeting agent linked to a targeted agent, such as a Renilla
reniformis GFP, a luciferase or luciferin. The systems also include
a second composition that contains the remaining components of a
bioluminescence generating reaction and/or the GFP. In some
embodiments, all components, except for activators, which are
provided in situ or are present in the body or tissue, are included
in a single composition.
[0522] In particular, the diagnostic systems include two
compositions. A first composition that contains conjugates that, in
preferred embodiments, include antibodies directed against tumor
antigens conjugated to a component of the bioluminescence
generating reaction, a luciferase or luciferin, preferably a
luciferase are provided. In certain embodiments, conjugates
containing tumor-specific targeting agents are linked to
luciferases or luciferins. In other embodiments, tumor-specific
targeting agents are linked to microcarriers that are coupled with,
preferably more than one of the bioluminescence generating
components, preferably more than one luciferase molecule.
[0523] The second composition contains the remaining components of
a bioluminescence generating system, typically the luciferin or
luciferase substrate. In some embodiments, these components,
particularly the luciferin are linked to a protein, such as a serum
albumin, or other protein carrier. The carrier and time release
formulations, permit systemically administered components to travel
to the targeted tissue without interaction with blood cell
components, such as hemoglobin that deactivates the luciferin or
luciferase.
[0524] 2. Methods of Diagnosing Diseases
[0525] Methods for diagnosing diseases, particularly infectious
diseases, using chip methodology, a luciferase/luciferin
bioluminescence-generating system, including a Gaussia, Pleuromamma
or Renilla mulleri luciferase plus a Renilla reniformis GFP, are
provided. In particular, the chip includes an integrated
photodetector that detects the photons emitted by the
bioluminescence-generating system as shifted by the GFP. This chip
device, which is described in copending U.S. application Ser. No.
08/990,103, which is published as International PCT application No.
WO 98/26277, includes an integrated photodetector that detects the
photons emitted by the bioluminescence generating system. The
method may be practiced with any suitable chip device, including
self-addressable and non-self addressable formats, that is modified
as described herein for detection of generated photons by the
bioluminescence generating systems. The chip device provided herein
is adaptable for use in an array format for the detection and
identification of infectious agents in biological specimens.
[0526] To prepare the chip, a suitable matrix for chip production
is selected, the chip is fabricated by suitably derivatizing the
matrix for linkage of macromolecules, and including linkage of
photodiodes, photomultipliers CCD (charge coupled device) or other
suitable detector, for measuring light production; attaching an
appropriate macromolecule, such as a biological molecule or
anti-ligand, e.g., a receptor, such as an antibody, to the chip,
preferably to an assigned location thereon. Photodiodes are
presently among the preferred detectors, and specified herein. It
is understood, however, that other suitable detectors may be
substituted therefor.
[0527] In one embodiment, the chip is made using an integrated
circuit with an array, such as an X-Y array, of photodetectors,
such as that described in co-pending U.S. application Ser. No.
08/990,103. The surface of circuit is treated to render it inert to
conditions of the diagnostic assays for which the chip is intended,
and is adapted, such as by derivatization for linking molecules,
such as antibodies. A selected antibody or panel of antibodies,
such as an antibody specific for particularly bacterial antigen, is
affixed to the surface of the chip above each photodetector. After
contacting the chip with a test sample, the chip is contacted with
a second antibody linked to the GFP, such as the Renilla GFP, to
form a chimeric antibody-GFP fusion protein or an antibody linked
to a component of a bioluminescence generating system, such as a
Pleuromamma, Gaussia or R. mulleri luciferase. The antibody is
specific for the antigen. The remaining components of the
bioluminescence generating reaction are added, and, if any of the
antibodies linked to a component of a bioluminescence generating
system are present on the chip, light will be generated and
detected by the adjacent photodetector. The photodetector is
operatively linked to a computer, which is programmed with
information identifying the linked antibodies, records the event,
and thereby identifies antigens present in the test sample.
[0528] 3. Methods for Generating Renilla mulleri Luciferase,
Pleuromamma Luciferase and Gaussia Luciferase Fusion Proteins with
Renilla reniformis GFP.
[0529] Methods for generating GFP and luciferase fusion proteins
are provided. The methods include linking DNA encoding a gene of
interest, or portion thereof, to DNA encoding a Renilla reniformis
GFP and a luciferase in the same translational reading frame. The
encoded-protein of interest may be linked in-frame to the amino- or
carboxyl-terminus of the GFP or luciferase. The DNA encoding the
chimeric protein is then linked in operable association with a
promoter element of a suitable expression vector. Alternatively,
the promoter element can be obtained directly from the targeted
gene of interest and the promoter-containing fragment linked
upstream from the GFP or luciferase coding sequence to produce
chimeric GFP proteins.
[0530] For example, a chimeric fusion containing the luciferase,
preferably a Renilla luciferase, more preferably a Renilla
reniformis luciferase, and Renilla reniformis GFP encoding DNA
linked to the N-terminal portion of a cellulose binding domain is
provided.
[0531] 4. Cell-Based Assays for Identifying Compounds
[0532] Methods for identifying compounds using recombinant cells
that express heterologous DNA encoding a Renilla reniformis GFP
under the control of a promoter element of a gene of interest are
provided. The recombinant cells can be used to identify compounds
or ligands that modulate the level of transcription from the
promoter of interest by measuring GFP-mediated fluorescence.
Recombinant cells expressing chimeric GFPs may also be used for
monitoring gene expression or protein trafficking, or determining
the cellular localization of the target protein by identifying
localized regions of GFP-mediated fluorescence within the
recombinant cell.
[0533] I. Kits
[0534] Kits may be prepared containing the Renilla reniformis GFP
or the encoding nucleic acid molecules (see, SEQ ID NOs. 23-26)
with or without components of a bioluminescence generating system
for use in diagnostic and immunoassay methods and with the novelty
items, including those described herein.
[0535] In one embodiment, the kits contain appropriate reagents and
an article of manufacture for generating bioluminescence in
combination with the article. These kits, for example, can be used
with a bubble-blowing or producing toy or with a squirt gun. These
kits can also include a reloading or charging cartridge.
[0536] In another embodiment, the kits are used for detecting and
visualizing neoplastic tissue and other tissues and include a first
composition that contains the Renilla reniformis GFP and a selected
luciferase, such as a Renilla mulleri, Renilla reniformis or
Gaussia luciferase, and a second that contains the activating
composition, which contains the remaining components of the
bioluminescence generating system and any necessary activating
agents.
[0537] In other embodiments, the kits are used for detecting and
identifying diseases, particularly infectious diseases, using
multi-well assay devices and include a multi-well assay device
containing a plurality of wells, each having an integrated
photodetector, to which an antibody or panel of antibodies specific
for one or more infectious agents are attached, and composition
containing a secondary antibody, such as an antibody specific for
the infectious agent that is linked, for example, to a Renilla
reniformis GFP protein, a chimeric antibody-Renilla reniformis GFP
fusion protein, F(Ab).sub.2 antibody fragment-Renilla reniformis
GFP fusion protein or to such conjugates containing the, for
example, Gaussia or Renilla mulleri or reniformis, luciferase. A
second composition containing the remaining components of a
bioluminescence generating system, such as system that emits a
wavelength of light within the excitation range of the GFP, such as
species of Renilla or Aequorea, for exciting the Renilla
luciferase, which produces green light that is detected by the
photodetector of the device to indicate the presence of the
agent.
[0538] In further embodiments, the kits contain the components of
the diagnostic systems. The kits comprise compositions containing
the conjugates, preferably Renilla GFP and a Gaussia, or
Pleuromamma or Renilla mulleri luciferase and remaining
bioluminescence generating system components. The first composition
in the kit typically contains the targeting agent conjugated to a
GFP or luciferase. The second composition, contains at least the
luciferin (substrate) and/or luciferase. Both compositions are
formulated for systemic, local or topical application to a mammal.
In alternative embodiments, the first composition contains the
luciferin linked to a targeting agent, and the second composition
contains the luciferase or the luciferase and a GFP.
[0539] In general, the packaging is non-reactive with the
compositions contained therein and where needed should exclude
water and or air to the degree those substances are required for
the luminescent reaction to proceed.
[0540] Diagnostic applications may require specific packaging. The
bioluminescence generating reagents may be provided in pellets,
encapsulated as micro or macro-capsules, linked to matrices,
preferably biocompatible, more preferably biodegradable matrices,
and included in or on articles of manufacture, or as mixtures in
chambers within an article of manufacture or in some other
configuration. For example, a composition containing luciferase
conjugate will be provided separately from, and for use with, a
separate composition containing a bioluminescence substrate and
bioluminescence activator.
[0541] Similarly, the Renilla reniformis GFP and selected
luciferase and/or luciferin, such as a Pleuromamma, Renilla mulleri
or Gaussia luciferase or luciferin, may be provided in a
composition that is a mixture, suspension, solution, powder, paste
or other suitable composition separately from or in combination
with the remaining components, but in the absence of an activating
component. Upon contacting the conjugate, which has been targeted
to a selected tissue, with this composition the reaction commences
and the tissue glows. In preferred embodiments, the tissue glows
green emitting light near 510 nm. The luciferase, GFP and
bioluminescence substrate, for example, are packaged to exclude
water and/or air, the bioluminescence activator. Upon
administration and release at the targeted site, the reaction with
salts or other components at the site, including air in the case of
surgical procedures, will activate the components. In some
embodiments, it is desirable to provide at least the GFPs or one
component of the bioluminescence generating system linked to a
matrix substrate, which can then be locally or systemically
administered.
[0542] Suitable dispensing and packaging apparatus and matrix
materials are known to those of skill in the art, and preferably
include all such apparatus described in U.S. patent Nos. see U.S.
Pat. Nos. 5,876,995, 6,152,358 and 6,113,886.
[0543] J. Muteins
[0544] Muteins of the Renilla reniformis GFP are provided herein.
Muteins in which conservative amino acid changes that do not alter
its ability to act as an acceptor of energy generated by a Renilla
luciferase/substrate reaction are provided. Also provided are
muteins with altered properties, including muteins with altered
spectral properties, muteins with altered surface properties that
reduce multimerization, including dimerization.
[0545] 1. Mutation of GFP Surfaces to Disrupt Multimerization
[0546] FIG. 5 depicts the three anthozoan fluorescent proteins for
which a crystal structure exists; another is available commercially
from Clontech as dsRed (also known as drFP583, as in this
alignment) (Wall et al. (2000); Nature Struct. Biol. 7:1133-1138;
Yarbrough et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:
462-467). A dark gray background depicts amino acid conservation,
and a light gray background depicts shared physiochemical
properties. These crystal structures and biochemical
characterization (Baird et al (2000) Proc. Natl. Acad. Sci. U.S.A.
97: 11984-11989) show that dsRed exists as a obligate tetramer in
vitro. Evidence also exists that dsRed multimerizes in living cells
(Baird et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:
11984-11989). Sedimentation and native gel electrophoresis studies
indicate that Ptilosarcus and Renilla mullerei GFPs also form
tetramers in vitro and multimerize in vivo. Ptilosarcus and Renilla
mullerei GFPs diverge strongly in amino acid sequence from dsRed
(39% and 38% identical, respectively). Computational polypeptide
threading algorithms predict that these GFPs fold into essentially
the same structure as dsRed, and also the much more sequence
divergent Aequorea victoria GFP. Renilla reniformis GFP is
similarly related in sequence to d/Red, Ptilosarcus and Renilla
mullerei GFPs (37%, 51% and 53% identical, respectively), and thus
is extremely likely to form similar multimers. Multimerization is
undesirable for many applications that use GFP as the reporting
moiety in chimeric protein fusion. Hence muteins in which the
capacity to multimerize is reduced are provided. Thus provided are
mutations Renia reniformis GFP that disrupt the formation of GFP
multimers. Such mutations may also be effected in the Ptilosarcus
and Renilla mullerei and other GFPs (see FIG. 6).
[0547] Two interaction surfaces within the dsRed tetramer, one
primarily hydrophobic (residues marked by X) and one primarily
hydrophilic (residues marked by O) have been described (see, Wall
et al. (2000); Nature Struct. Biol. 7:1133-1138). In general, the
corresponding residues vary considerably between the 4 GFPs in a
complex way, although the physicochemical properties of the amino
acids are often conserved. There are a few clusters of conserved
residues, especially between Ptilosarcus and Renilla mullerei GFPs,
in keeping with their 77% overall identity.
[0548] The scheme provided herein for disruption focuses on
altering surface amino acid side chains so that the surfaces
acquire or retain a hydrophilic character, and are also altered in
their stereo-chemistry (the sizes of the side chains are altered).
These GFP surface regions roughly map to the .beta.-sheet secondary
structures that comprise the GFP .beta.-barrel tertiary structure.
It is thus essential that the secondary structure in any surface
mutants be retained, so that the choice of amino acid side chain
substitutions is governed by this consideration.
[0549] It is also desirable to introduce mutations that alter
charge. For example, such mutations are those in which R, H and K
residues have been replaced with D, such that the hydrophobic and
hydrophilic surfaces now each contain 3 mutated residues (SEQ ID
NO.33; Lys to Asp at amino acids 108, 127 and 226, Arg to Asp at
amino acids 131 and 199; His to Asp at amino acid 172.
[0550] Site directed mutagenesis techniques are used to introduce
amino acid side chains that are amenable to aqueous solvation, and
that significantly alter surface sterochemistry. Disruption of
interacting surfaces involves loss-of-function mutagenesis. It is
thus contemplated that altering only a few residues, perhaps even
one, is sufficient.
[0551] 2. Use of Advantageous GFP Surfaces with Substituted
Fluorophores
[0552] Other surfaces of GFPs may be key determinants of GFP
usefulness as reporters in living systems. A GFP surface may
adventitiously interact with vital cellular components, thereby
contributing to GFP-induced cytoxicity. Anthozoan GFPs from
bioluminescent luciferase-GFP systems serve fundamentally different
biological functions than do anthozoan GFPs from coral and
anemones. The Renilla reniformis GFP is present in low quantity and
functions as a resonance energy acceptor in response to a dynamic
neural network that enables a startled animal to emit light
flashes. A coral GFP-like protein is present in large quantity and
apparently is used primarily as a passive pigment; it may not have
evolved to dynamically interact with sensitive cellular machinery.
These two classes of anthozoan fluorescent proteins thus may have
surfaces with markedly different biological properties.
[0553] FIG. 4 exemplifies the site for substitution for inserting
fluorophores into the background of Ptilosarcus, Renilla mullerei
and Renilla reniformis GFPs. In particular, the 20 amino acid
region that lies between two highly conserved prolines with the
corresponding 20 amino acid region from any other anthozoan GFP
(the underlined regions corresponds to amino acids 56-75 of SEQ ID
NO. 27 Renilla reniformis GFP; amino acids 59-78 of SEQ ID NO. 16
Renilla mulleri GFP; and amino acids 59-78 of SEQ ID NO. 32 for
Ptilosarcus GFP) is replaced or modified. These 20 residues
comprise the bulk of a polypeptide region that threads along the
interior of the .beta.-barrel structure that is characteristic of
anthozoan GFPs (Wall et al. (2000) Nature Struct. Biol.
7:1133-1138; Yarbrough et al. (2001) Proc. Natl. Acad. Sci. U.S.A.
98: 462-467); replacement or modification alters spectral
properties.
[0554] K. Transgenic Plants and Animals
[0555] As discussed above, transgenic animals and plants that
contain the nucleic acid encoding the Renilla reniformis GFP are
provided. Methods for producing transgenic plants and animals that
express a GFP are known (see, e.g., U.S. Pat. No. 6,020,538).
[0556] Among the transgenic plants and animals provided are those
that are novelty items, such as animals with eyes or fingernails or
tusks or hair that glows fluorescently. Transgenic food animals,
such as chickens and cows and pigs are contemplated from which
glowing meat and eggs (green eggs and ham) can be obtained; glowing
worms can serve as fishing lures. In addition, the Renilla
reniformis can serve as a reporter to detect that a heterologous
gene linked to the GFP gene is incorporated into the animal's
genome or becomes part of the genome in some or all cells. The
Renilla reniformis can similarly be used as a reporter for gene
therapy. The GFP can be introduced into plants to make transgenic
ornamental plants that glow, such as orchids and roses and other
flowering plants. Also the GFP can be used as a marker in plants,
such as by linking it to a promoter, such as Fos that responds to
secondary messages to assess signal transduction. The GFP can be
linked to adenylcyclase causing the plants to emit different
spectral frequencies as the levels of adenylcyclase change.
[0557] L. Bioluminescence Resonance Energy Transfer (BRET)
System
[0558] In nature, coelenterazine-using luciferases emit broadband
blue-green light (max. .about.480 nm). Bioluminescence Resonance
Energy Transfer (BRET) is a natural phenomenon first inferred from
studies of the hydrozoan Obelia (Morin & Hastings (1971) J.
Cell Physiol. 77:313-18), whereby the green bioluminescent emission
observed in vivo was shown to be the result of the luciferase
non-radiatively transferring energy to an accessory green
fluorescent protein (GFP). BRET was soon thereafter observed in the
hydrozoan Aequorea victoria and the anthozoan Renilla reniforms.
Although energy transfer in vitro between purified luciferase and
GFP has been demonstrated in Aequorea (Morise et al. (1974)
Biochemistry 13:2656-62) and Renilla (Ward & Cormier (1976) J.
Phys. Chem. 80:2289-91) systems, a key difference is that in
solution efficient radiationless energy transfer occurs only in
Renilla, apparently due to the pre-association of one luciferase
molecule with one GFP homodimer (Ward & Cormier (1978)
Photochem. Photobiol. 27:389-96). The blue (486 nm) luminescent
emission of Renilla luciferase can be completely converted to
narrow band green emission (508 nm) upon addition of proper amounts
of Renilla GFP (Ward & Cormier (1976) J. Phys. Chem.
80:2289-91). GFPs accept energy from excited states of
luciferase-substrate complexes and re-emit the light as narrow-band
green light (.about.510 nm). By virtue of the non-radiative energy
transfer, the quantum yield of the luciferase is increased.
[0559] Luciferases and fluorescent proteins have many
well-developed and valuable uses as protein tags and
transcriptional reporters; BRET increases the sensitivity and scope
of these applications. A GFP increases the sensitivity of the
luciferase reporter by raising the quantum yield. A single
luciferase fused (or chemically linked) to several spectrally
distinct GFPs provides for the simultaneous use of multiple
luciferase reporters, activated by addition of a single luciferin.
By preparing two fusion proteins (or chemical conjugates), each
containing a GFP having a different emission wavelength fused to
identical luciferases, two or more reporters can be used with a
single substrate addition. Thus multiple events may be monitored or
multiple assays run using a single reagent addition. Such a
reporter system is self-ratioing if the distribution of luciferin
is uniform or reproducible.
[0560] The ability to conveniently monitor several simultaneous
macromolecular events within a cell is a major improvement over
current bioluminescent technology. BRET also enables completely new
modes of reporting by exploiting changes in association or
orientation of the luciferase and fluorescent protein. By making
fusion proteins, the luciferase-GFP acceptor pair may be made to
respond to changes in association or conformration of the fused
moieties and hence serves as a sensor.
[0561] Energy transfer between two fluorescent proteins (FRET) as a
physiological reporter has been reported (Miyawaki et al. (1997)
Nature 388:882-7), in which two different GFPs were fused to the
carboxyl and amino termini of calmodulin. Changes in calcium ion
concentration caused a sufficient conformational change in
calmodulin to alter the level of energy transfer between the GFP
moieties. The observed change in donor emission was .about.10%
while the change in ratio was .about.1.8.
[0562] FIG. 2, reproduced from allowed copending application U.S.
application Ser. No. 09/277,716, illustrates the underlying
principle of Bioluminescent Resonance Energy Transfer (BRET) using
GFPs and luciferase, preferably cognate luciferase, and its use as
sensor: A) in isolation, a luciferase, preferably an anthozoan
luciferase, emits blue light from the coelenterazine-derived
chromophore; B) in isolation, a GFP, preferably an anthozoan GFP
that binds to the luciferase, that is excited with blue-green light
emits green light from its integral peptide based fluorophore; C)
when the luciferase and GFP associate as a complex in vivo or in
vitro, the luciferase non-radiatively transfers its reaction energy
to the GFP fluorophore, which then emits the green light; D) any
molecular interaction that disrupts the luciferase-GFP complex can
be quantitatively monitored by observing the spectral shift from
green to blue light. Hence, the interaction or disruption thereof
serves as a sensor.
[0563] The similar use of a luciferase-GFP pair in the presence of
substrate luciferin has important advantages. First, there is no
background and no excitation of the acceptor from the primary
exciting light. Second, because the quantum yield of the luciferase
is greatly enhanced by nonradiative transfer to GFP, background
from donor emission is less, and the signal from the acceptor
relatively greater. Third, the wavelength shift from the peak
emission of luciferase (.about.480 nm) to that of the GFP
(typically 508-510 nm) is large, minimizing signal overlap. All
three factors combine to increase the signal-to-noise ratio. The
concentration of the GFP acceptor can be independently ascertained
by using fluorescence.
[0564] For some applications, in vitro crosslinked or otherwise in
vitro modified versions of the native proteins is contemplated. The
genetically encoded fusion proteins have many great advantages: A)
In vivo use--unlike chemistry-based luminescence or
radioactivity-based assays, fusion proteins can be genetically
incorporated into living cells or whole organisms. This greatly
increases the range of possible applications; B) Flexible and
precise modification--many different response modifying elements
can be reproducibly and quantitatively incorporated into a given
luciferase-GFP pair; C) Simple purification--only one reagent would
need to be purified, and its purification could be monitored via
the fluorescent protein moiety. Ligand-binding motifs can be
incorporated to facilitate affinity purification methods.
[0565] 1. Design of Sensors Based on BRET
[0566] Resonance energy transfer between two chromophores is a
quantum mechanical process that is exquisitely sensitive to the
distance between the donor and acceptor chromophores and their
relative orientation in space (Wu & Brand (1994) Anal. Biochem.
218:1-13). Efficiency of energy transfer is inversely proportional
to the 6.sup.th power of chromophore separation. In practice, the
useful distance range is about 10 to 100 A, which has made
resonance energy transfer a very useful technique for studying the
interactions of biological macromolecules. A variety of
fluorescence-based FRET biosensors have been constructed, initially
employing chemical fluors conjugated to proteins or membrane
components, and more recently, using pairs of spectrally distinct
GFP mutants (Giuliano & Taylor (1998) Trends Biotech.
16:99-146; Tsien (1998) Annu. Rev. Biochem. 67:509-44).
[0567] Although these genetically encoded GFP bioluminescence-based
biosensors have advantages over less convenient and less precise
chemical conjugate-based biosensors, all share a limitation in
their design: it is generally difficult to construct a biosensor in
which energy transfer is quantitative when the chromophores are in
closest apposition. It is almost impossible to arbitrarily
manipulate the complex stereochemistry of proteins so that
conjugated or intrinsic chromophores are stably positioned with
minimal separation and optimal orientation. The efficiency of such
biosensors are also often limited by stoichiometric imbalances
between resonance energy donor and acceptor; the donor and acceptor
macromolecules must be quantitatively complexed to avoid background
signal emanating from uncomplexed chromophores. These limitations
in general design become important when biosensors must be robust,
convenient and cheap. Developing technologies such as high
throughput screening for candidate drugs (using high throughput
screening (HTS) protocols), biochips and environmental monitoring
systems would benefit greatly from modular biosensors where the
signal of a rare target "hit" (e.g., complex formation between two
polypeptides) is unambiguously (statistically) distinguishable from
the huge excess of "non-hits"). Current genetically encoded FRET
and bioluminescence-based biosensors display hit signals that very
often are less than two-fold greater than non-hit signals, and are
at best a few-fold greater (Xu et al. (1999) Proc. Natl. Acad. Sci
USA 96: 151-156; Miyawaki et al. (1997) Nature 388:882-7).
[0568] To solve these problems, the anthozoan GFPs, particularly
the Renilla GFPs, provided herein can be used in combination with
its cognate luciferases. Anthozoan luciferases -GFP complexes
provide a "scaffold" upon which protein domains that confer the
biological properties specific to a given biosensor can be linked.
Although one can construct many useful two component biosensors
based on this scaffold, in a biosensor contemplated herein,
independent protein domains that potentially complex with one
another are respectively fused to the luciferase and the GFP.
[0569] There are many possible variations on this theme. For
example, in a three component system either the luciferase or GFP
can be fused to a ligand-binding domain from a protein of interest
or other target peptide or other moiety of interest. If the design
of the fusion protein is correct, binding of a small molecule or
protein ligand then prevents the luciferase-GFP association. The
resulting combination of elements is a BRET-based biosensor; the
change in spectral properties in the presence and absence of the
ligand serves as sensor. More complex protein fusions can be
designed to create two component and even single component BRET
biosensors for a multitude of uses.
[0570] The nucleic acids, and the constructs and plasmids herein,
permit preparation of a variety of configurations of fusion
proteins that include an anthozoan GFP, in this case Renilla
reniformis, preferably with a Renilla luciferase, more preferably
with the Renilla reniformis luciferase. The nucleic acid encoding
the GFP can be fused adjacent to the nucleic acid encoding the
luciferase or separated therefrom by insertion of nucleic acid
encoding, for example, a ligand-binding domain of a protein of
interest. The GFP and luciferase will be bound. Upon interaction of
the ligand-binding domain with the a test compound or other moiety,
the interaction of the GFP and luciferase will be altered thereby
changing the emission signal of the complex. If necessary the GFP
and luciferase can be modified to fine tune the interaction to make
it more sensitive to conformational changes or to temperature or
other parameters.
[0571] 2. BRET Sensor Architectures
[0572] FIG. 3 depicts some exemplary BRET sensor architectures. The
upper left panel depicts the luciferase-GFP scaffold, the basis for
the representative BRET sensor architectures shown here. The
depicted single polypeptide fusion constructs place the luciferase
and GFP at the polypeptide termini, bracketing interacting protein
domains of choice. The luciferase and GFP can alternatively be
placed centrally within the polypeptide, between interacting
protein domains (not shown). This alternative arrangement is
advantageous for one step protein interaction-based cloning
schemes, where cDNA fragments encoding potential protein targets
can be ligated onto one end of the construct.
[0573] Single polypeptide sensors that detect conformational
changes within protein targets or the association-dissociation of
protein targets are well-suited for the detection of physiological
signals, such as those mediated by phosphorylation or other
modification of targets, or by binding of regulatory ligands, such
as hormones, to targets. Sensors based on interference are best
suited to assaying the presence of small molecules or proteins
independent of any regulatory context. Quantitative assays of
metabolites, such as a sugar and allergens, are among those
contemplated.
[0574] Since in vivo and in vitro luciferase-to-GFP energy transfer
can be nearly 100% efficient, binding interactions between the
luciferase and GFP must be sufficient to establish an optimal
spatial relationship between donor and acceptor chromophores.
Optimization of the luciferase-GFP energy transfer module is
important in building effective BRET sensors. In a single
polypeptide sensor it is crucial that the luciferase-GFP
interaction be weak relative to interactions between target
domains, thus the need for an optimized energy transfer module. In
practice, either the luciferase or GFP surface can be randomly
mutagenized, and an optimized luciferase-GFP scaffold then selected
by screening for either blue or green emission at two near
physiological temperatures (thermal endpoint-selection) using
current robotic systems. This disruption of BRET is readily
achievable because loss-of-function mutants (weakened
luciferase-GFP binding) are orders of magnitude more frequent than
gain-of-function mutants.
[0575] With an optimized energy transfer scaffold in hand, thermal
endpoint-selection can then be used, if necessary, to optimize the
interactions between the target domains incorporated into a sensor.
This second round of thermal endpoint-selection may be especially
important for the construction of interference sensors because it
is essential that such sensors be able to "open and close" at near
physiological temperatures to sense interference. Thermal
endpoint-selection can also be used to weaken the binding affinity
of the analyte to the interference sensor, making it possible to
thermally wash off the analyte and reuse the sensor, a great
advantage for biochip-based applications.
[0576] 3. Advantages of BRET Sensors
[0577] There are many advantages to the BRET sensors provided
herein. For example, BRET sensors are self-ratioing. The reporter
and target are integrated into single polypeptide. This ensures
1:1:1 stoichiometry among luciferase, GFP and target (or a 1:N:1
stoichiometry if more than one, typically a homodimer, GFP can be
bound to a luciferase). GFP fluorescence allows absolute
quantitation of sensor. The null state gives signal that verifies
sensor functionality. Quantifiable null state facilitates
disruption-of-BRET sensors (DBRET). BRET sensors have better
signal-to-noise ratio than GFP FRET sensors because there is no
cellular autofluorescence, no excitation of the acceptor from the
primary exciting light, the quantum yield of luciferase greatly
enhanced by non-radiative energy transfer to GFP, and there is
minimal signal overlap between emission of the luciferase and
emission of the GFP. Also, anthozoan GFPs have 6-fold higher
extinction coefficients than Aequorea GFP.
[0578] The BRET sensors can be used for hit identification and
downstream evaluation in in vitro screening assays in in vitro or
in vivo or in situ, including in cultured cells and tissues and
animals. The BRET sensors can be created by thermal
endpoint-selection, which is suited to DBRET (Disruption-of-BRET)
and reduces need for knowledge of target 3D structure and
functional dynamics. Existing screening robotics can be used to
optimize biosensors. BRET sensors benefit from vast genetic
diversity anthozoans have evolved efficient luciferase-GFP energy
transfer systems and the components can be mixed and matched.
Highly efficient heterologous luciferases may be substituted for
less active luciferases. For example, a copepod luciferase active
site can be fused to an anthozoan luciferase GFP-binding domain.
There are many diverse coelenterazine-using luciferases.
[0579] BRET sensors are modular so that an optimized sensor
scaffold may be used with different targets. Also the BRET acceptor
may be varied to give shifted emissions, facilitating multiple
simultaneous readouts. The anthozoan GFPs can be mutated, GFPs or
other proteins can be modified with different chemical fluors, high
throughput screening (HTS) fluor-modified FRET acceptors can be
adapted, and the BRET donor (luciferase) may be varied, such as by
using an Aequorin (Ca++ activated) photoprotein, or a firefly
luciferse (requires ATP and a firefly luciferin) to give
conditional activation. The sensor scaffold can be incorporated
into a variety of immobilization motifs, including free format
plates, which can reduce reagent volumes, reusable microtiter
plates, miniature columns and biochips. Finally, BRET sensors are
inexpensive and reproducible reagents because they can be produced
by standardized protein production and can incorporate purification
tags. Genetically encoded reporters more reproducible than
chemically modified reporters. Linear translation of BRET modules
ensures sensor integrity.
[0580] The following example is included for illustrative purposes
only and is not intended to limit the scope of the invention.
EXAMPLE
[0581] Specimens of the sea pansy Renilla reniformis were collected
from inshore waters off the coast of Georgia. To prepare the sea
pansies for isolation of mRNA, about 25 or so at time were placed
on a large bed of dry ice. They were flipped with a spatula to flip
them over to prevent them from freezing. Oddly, the entire animal
illuminated when it came in contact with the dry ice. The brightest
and greenest were culled, placed in a bag and back into sea water
at about 65-70.degree. C. for two hours. This process of dry ice,
culling and sea water treatment was repeated three times over a 6
hour period. In addition, the process was performed at night. After
they were exhausted with the last chilling, the culled animals were
frozen solid. A cDNA library was prepared from the frozen
animals.
[0582] The animals that were selected this way were frozen in
liquid nitrogen, and shipped to Stratagene, Inc. (La Jolla,
Calif.), a commercial vendor whose business includes the
construction of custom cDNA libraries under contract to prepare the
library. Purified polyA-mRNA was prepared, and a cDNA synthesis
reaction was performed, appending a 3' XhoI site and a 5' EcoRI
restriction site to the cDNA. The cDNA was inserted by ligation
between the EcoRI and XhoI sites of the Uni-ZAP Lambda phage cDNA
cloning vector.
[0583] The resulting unamplified library contained approximately
1.6.times.10.sup.8 primary plaques, which after amplification gave
a titer of 3.5-7.5 pfb (plaque forming units)/ml. Insert sizes
ranged from 0.9 to 3.0 kb, with an average size around 1.5 kb. Two
mass excisions were performed to give pBluescript phagemid
containing the cDNA inserts; each excision from about 8.times.10
plaques gave rise to about 4.8.times.10.sup.9 cfu (colony forming
units)/ml. Phagemids were transfected into the SOLR strain of E.
coli.
[0584] Screening was performed by plating (using an artist's
airbrush) approximately 200,000 colonies to each of 40 cafeteria
trays containing LB agar medium incorporating 0.4% carbon black to
absorb background fluorescence. After 24 hours growth at 30.degree.
C. in a humidified incubator, GFP expressing colonies were
identified by illuminating the plates using a 250 Watt quartz
halogen fiber optics light (Cuda Products Corp) with an EGFP
bandpass excitation filter (Chroma), and viewing colonies through a
GFP bandpass emission filter. Approximately 10 fluorescent colonies
were picked, DNA isolated from minipreps, and the DNA transformed
into the XL-10 Gold E. coli strain (Stratagene). Analysis by
restriction digestion resolved three distinguishable sizes of
insert. DNA was prepared from a clone of each size class and sent
to SeqWright LLB (Houston, Tex.) for sequencing. Sequencing data
were reported to Prolume on 1-25-99.
[0585] Three independent cDNA clones of Renilla reniformis GFP were
isolated (SEQ ID NOs 23-25). Each cDNA is full length as judged by
identical 5' termini and each encodes an identical protein of 233
amino acids (see SEQ ID NO. 27). Compared to the primary clone
(Clone 1), the coding sequence of Clone 2 differs by 4 silent
mutations. Clones 2 and 3 also contain small differences in the 5'
and 3' untranslated regions of the cDNA. This nucleic acid has been
inserted into expression vector, and the encoded protein
produced.
[0586] Since modifications may be apparent to those of skill in the
art, it is intended that the invention be limited only by the
appended claims.
Sequence CWU 0
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