WO2005116219A1 - Fluorescent protein - Google Patents

Abstract

The invention relates to a fluorescent protein which is useful as a Ca2+ indicator, as well as nucleic acids and expression vectors encoding such a protein and host cells expressing such a protein. The invention further relates to methods of using the fluorescent protein to measure cellular Ca2+ levels and to cell-based assays using the fluorescent protein for detecting Ca2+ levels. The invention further relates to screening methods using the fluorescent protein. In one aspect, the fluorescent protein is targeted to the endoplasmic reticulum (ER) for the measurement of Ca2+ levels in the ER.

Description

Fluorescent Protein

Field of the invention

The present invention relates to a fluorescent protein which is useful as a Ca²⁺ indicator. In particular, the present invention relates to a fluorescent protein useful as an indicator of Ca²⁺ at levels found in the endoplasmic reticulum (ER) of a cell .

Background of the invention

Fluorescent proteins for use as indicators of Ca²⁺ levels within a single living cell are known. In particular, fluorescent proteins have been developed for the determination of Ca²⁺ levels in specific compartments of a cell, such as the cytosol , nucleus and ER.

EP 1 238 982 A describes the construction of a range of fluorescent proteins which are sensitive to Ca²⁺ and can be used as indicators of Ca²⁺ levels. A yellow fluorescent protein (YFP) variant (EYFP) having mutations at positions 68 and 69 of the amino acid sequence (V68L/Q69K) was subjected to circular permutation in which the N and C termini were interchanged and reconnected by a short spacer between the original termini. Thus, the original N and C termini were linked through a short linker to give Y145 and N144 as the new N and C termini, respectively, creating cpEYFP. Then, cpEYFP was fused to the C terminus of M13, a target peptide of calmodulin, and to the N terminus of calmodulin, a Ca²⁺-binding protein (CaM) . The resulting protein, named "pericam" was both fluorescent and showed

Ca²⁺ sensitivity. Ca²⁺-bound calmodulin and M13 are known to form a stable and compact complex. The fluorescence characteristic of pericam varies according to the Ca²⁺- dependent interaction between calmodulin and M13.

Variants of pericam were obtained by mutating various residues near to the chromophore . Of these pericam variants, "flash-pericam" exhibited increased fluorescence in the presence of Ca²⁺, whereas "inverse- pericam" exhibited decreased fluorescence in the presence of Ca²⁺. Unlike these two variants, "ratiometric-pericam" has a dual wavelength excitation spectrum such that it permits quantitative Ca²⁺ imaging.

A further Ca²⁺-sensitive fluorescent protein has been described (Baird G S et al . (1999) Proc Natl Acad Sci USA 96, 11241-11246). The protein, named "camgaroo", is an enhanced YFP in which calmodulin has been inserted in the place of Y145. This protein has been used to measure cytosolic Ca²⁺ levels in single mammalian cells.

Further, a fluorescent reporter protein for the ER has been described, based on "cameleon" fluorescent indicators (Miyawaki A et al . (1997) Nature 388, 882- 887) . These cameleon indicators consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP) , calmodulin, the calmodulin- binding peptide Ml3 , and an enhanced green- or yellow- emitting GFP mutant. Binding of Ca²⁺ to the calmodulin causes the calmodulin to interact with the Ml3 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs . Free Ca²⁺ concentrations have been visualised in the ER of single

HeLa cells transfected with a cDNA encoding a cameleon chimaera having an appropriate ER localisation signal.

Thus, cameleon is a dual -emission ratiometric Ca²⁺ indicator that employs FRET with two different GFP mutants of different colour.

However, the cameleon indicator has a low signal-to-noise ratio in vi tro indicating that it is unlikely to provide clear, functional data if used in vivo . Further, this indicator is sensitive to pH with the consequence that it will not work efficiently in the ER. Additionally, the FRET based method of measurement requires measurement at two separate emission wavelengths which is difficult to detect and interpret due to overlapping and irregular spectra of the fluorescent proteins. As such, cameleon is limited in its usefulness as an indicator of ER Ca²⁺ levels .

The pericams of EP 1 238 982 A have been used to measure free Ca²⁺ levels in the cytosol and nucleus. Ratiometric- pericam has additionally been used to measure mitochondrial Ca²⁺ concentrations. However, none of these pericams have been used to measure Ca²⁺ levels in the ER. Indeed, these pericams are not suitable for measuring ER Ca²⁺ levels. This is predominantly due to their sensitivity to Ca²⁺. For example, flash-, ratio-metric- and inverse-pericam all have reasonably high affinities for Ca²⁺ (reported iS = 0.7 μM, 1.7 μM and 0.2 μM, respectively, EP 1 238 982 A, Table 1) , making them unsuitable for detecting the relatively high Ca²⁺ concentrations found in the ER compared to the cytosol.

Therefore, there is a need for Ca²⁺ indicators that are suitable for determining Ca²⁺ at levels found in the ER and may be used to determine Ca²⁺ levels in the ER. A suitable indicator for this purpose should have a sensitivity for Ca²⁺ that allows reliable indication of the physiological levels of Ca²⁺ found in the ER, which are relatively high compared to certain other compartments of the cell. Further, the indicator should be able to be provided with a suitable ER localization signal such that it is retained in the ER inside the cell.

Summary of the invention

The present invention aims to address the need for an indicator for determining Ca²⁺ at levels found in the ER of a single living cell.

Generally, the invention lies in providing a fluorescent protein that has appropriate sensitivity to Ca²⁺ having regard to the physiological concentrations of Ca²⁺ found in the ER such that it is suitable for use in the determination of Ca²⁺ levels in the ER. Such a fluorescent protein is also suitable for determining Ca²⁺ levels in a cell compartment having similar Ca²⁺ levels to those found in the ER, for example, the sarcoplasmic reticulum (SR) .

Thus, generally, the invention lies in providing a fluorescent protein having altered sensitivity to Ca²⁺, involving modification of the Ca²⁺-binding ability of the protein. The invention is further concerned with modifying residues in a Ca²⁺-binding domain of a fluorescent protein. The invention relates to modifying residues of calmodulin in general, and residues of a calmodulin domain of a fluorescent protein in particular. More specifically, an EF hand of calmodulin is the target domain for modification. The invention further relates to fluorescent proteins having reduced sensitivity to Ca²⁺. Furthermore, the invention is concerned with maintaining the Ca²⁺-dependent fluorescent properties of a fluorescent protein. In general, the invention relates to the structural changes a fluorescent protein undergoes on binding Ca²⁺ and in particular, those structural changes involved in ensuring fluorophore formation in the presence of Ca²⁺. The invention further relates to fluorescent proteins having restored fluorescent properties. The invention is further concerned with modifying residues of a fluorescent protein to ensure fluorophore formation in the presence of Ca²⁺.

Given the inherent disadvantages of the cameleon indicator described above, it was decided to attempt to adapt two of the more recently developed species of fluorescent proteins that are sensitive to Ca²⁺ to produce a novel fluorescent protein suitable for determining Ca²⁺ levels found in the ER. Thus, the fluorescent proteins chosen as targets for adaptation for use for determining Ca²⁺ levels in the ER were the pericam proteins (EP 1 238 982 A) and camgaroo protein (Baird G S et al., 1999) . In particular, the fluorescent protein ratiometric-pericam was chosen as a starting point given that, in addition to fluorescing in a Ca²⁺-dependent manner, it has a dual wavelength excitation spectrum such that it permits quantitative Ca²⁺ measurements.

Accordingly, in a first aspect, the present invention provides a fluorescent protein comprising in the direction from the N terminus to the C terminus (a) an amino acid sequence of a target peptide of calmodulin; (b) an amino acid sequence from the n^th amino acid from the N terminus to the C terminus of a fluorescent protein where n represents an integer of 140 to 148, wherein the amino acid sequence is modified by substitution of the amino acid at position 148; (c) a linker sequence of 2 to 20 amino acids; (d) an amino acid sequence from the first amino acid to the (n-l)^th amino acid from the N terminus of the fluorescent protein in (b) ; (e) the amino acid sequence of calmodulin, wherein the amino acid sequence is modified by substitution of one or more of the conserved bidentate glutamic acid residues at the 12^th residue of the first, second or fourth EF hand of calmodulin; and wherein the fluorescent protein can emit fluorescence that is dependent on Ca²⁺ concentration.

Thus, the present invention provides a novel fluorescent protein comprising calmodulin target peptide, fluorecent protein, linker, fluorescent protein and calmodulin domains (corresponding to domains (a) to (e) , respectively) , which is adapted from the fluorescent pericam proteins of EP 1 238 982 A, but which fluoresces in a Ca²⁺-dependent manner. Embodiments of the fluorescent protein of the invention have an altered Ca⁺- sensitivity as a result of modifications made to the calmodulin amino acid sequence. Thus, as a result, the invention provides a fluorescent protein that has a Ca²⁺- sensitivity suitable for use in determining Ca²⁺ concentrations found in the ER. Such a fluorescent protein may be used for determining Ca²⁺ levels in the ER and in other cell compartments having similar physiological Ca²⁺ levels as found in the ER.

Binding of Ca²⁺ to calmodulin causes calmodulin to bind more tightly to the M13 peptide. In a fluorescent protein such as ratiometric-pericam, this changes the structure surrounding the chromophore resulting in a fluorescence emission intensity change. Calmodulin has four Ca²⁺-binding domains known as EF hands . Each of these four EF hands has a conserved bidentate glutamic acid at the 12^th residue of the EF hand. Modification of any of these glutamic acid residues affects the strength of the binding of calmodulin to Ca²⁺. Thus, the substitution of one or more of these glutamic acid residues for a different amino acid affects the binding of the fluorescent protein to Ca²⁺ and thus the sensitivity of the protein to Ca²⁺ .

A consequence of present modifications of one or more of the calmodulin Ca²⁺-binding domains in ratiometric-pericam was that the fluorescent properties of the protein were lost . A strategy to restore fluorescence by making linkers in the protein more flexible, for example by including extra glycine residues, was unsuccessful. Inverse-pericam is identical in structure to ratiometric- pericam except for a single amino acid substitution at position 148 in the mutant YFP domain. This difference has been shown to cause the protein to exhibit decreased fluorescence in the presence of Ca²⁺ (EP 1 238 982 A) . However, the present applicant has found that modifying the amino acid at position 148 of the fluorescent protein domain in combination with modification of one or more of the Ca²⁺-binding sites of the calmodulin domain of the protein has the surprising effect of restoring fluorescence to the protein. Thus, a further substitution is required at position 148 in order to restore fluorescence to generate a fluorescent protein of the invention.

A "fluorescent protein" for amino acid sequences (b) and (d) is defined as including green fluorescent protein (GFP) and its mutants and all known fluorescent proteins derived therefrom and mutants thereof. For example, the gene for green fluorescent protein has been isolated and its sequence has been determined (Prasher D C et al . (1992) Gene 111, 229-233) . Thus, the sequences of GFP and its mutants are well known in the art (for example, see Tsin (1998) Ann. Rev. Biochem. 67, 509-544) . For example, yellow fluorescent protein (YFP) is a mutant of GFP having a T203Y substitution (Ormό et al . (1996) Science 273, 1392-1395). A variant of YFP, termed EYFP, has the further substitutions V68L and Q96K (Miyawaki et al. (1999) Proc. Natl . Acad. Sci . USA 96, 2135-2140).

The amino acid sequences (b) and (d) of such a fluorescent protein may be that of a fluorescent protein mutant having the substitutions V68L, Q69K and Y203F, where the first single-letter code is for the amino acid being replaced, followed by its numerical position in the sequence, and then the single-letter code for the replacement amino acid.

Preferably, in amino acid sequences (b) and (d) , n is 145.

"Calmodulin" is defined as including any known calmodulin, for example the calmodulin from Xenopus . Calmodulin is well conserved between organisms. In particular, calmodulin has four conserved Ca²⁺-binding domains known as EF hands . In each of these four EF hands there is a conserved bidentate glutamic acid residue at the 12^th residue of the EF hand. In Xenopus calmodulin, this corresponds to the glutamic acid residue at each of positions 31, 67, 104 and 140 of the calmodulin amino acid sequence, respectively. A "target peptide of calmodulin" is defined as including any peptide which has the amino acid sequence of a calmodulin-binding domain, which is known to exist in various proteins and peptides known to be targets of calmodulin. Currently, over 1200 types of amino acid sequence of calmodulin-binding domains are known. Preferably, the target peptide is the M13 peptide which is a 26-residue peptide derived from the calmodulin- binding region of the skeletal muscle myosin light-chain kinase (Blumenthal D K and Krebs E G (1987) Methods Enzymol . 139, 115-126). Alternatively, the target peptide may be from the calmodulin-dependent kinase kinase (CKKp) (Osawa M et al . (1999) Nat Struct Biol 6 (9) , 819-824) .

"Substitution" of an amino acid describes a modification where an amino acid at a particular position is replaced by a non-identical amino acid. The substitution may be a conservative substitution, in which an amino acid is replaced with one having similar properties, or a non- conservative substitution in which the replacement amino acid has different properties. "Addition" and "deletion" of an amino acid describe a modification where one or more amino acids are added to, or deleted at a particular position in an amino acid sequence .

Similarly, "substitution", "addition" and "deletion" of a nucleotide describes a modification where one or more nucleotides are replaced, added or removed at a particular position in a nucleic acid. The number of residues modified is not limited provided that the resultant fluorescent protein encoded by the nucleic acid has the ability to emit fluorescence that is dependent on Ca²⁺ concentration.

A fluorescent protein of the invention can emit fluorescence that is dependent on Ca²⁺ concentration. Preferably, a fluorescent protein of the invention has an altered Ca²⁺ sensitivity as a result of modifications made to the calmodulin amino acid sequence, compared to that provided by wild type calmodulin. More preferably, a fluorescent protein of the invention has a reduced sensitivity to Ca²⁺ compared to that provided by wild type calmodulin.

Preferably, modification of amino acid sequence (b) is by substitution of the amino acid at position 148 with threonine .

Amino acid sequence (e) may be modified by substitution of the conserved glutamic acid residue at the 12^th residue of the second EF hand of calmodulin. Where the calmodulin is from Xenopus, this corresponds to position E67 in the calmodulin amino acid sequence. Preferably, amino acid sequence (e) is modified by substitution of the conserved glutamic acid residue at the 12^th residue of the fourth EF hand of calmodulin. Where the calmodulin is from Xenopus, this corresponds to position E140 in the calmodulin amino acid sequence. More preferably, amino acid sequence (e) is modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first EF hand of calmodulin. In this case, where the calmodulin is from Xenopus, this corresponds to position E31 in the calmodulin amino acid sequence. Preferably, modification of amino acid sequence (e) by substitution of one or more of the conserved glutamic acid residues at the 12^th residue of the first, second or fourth EF hand is by substitution with glutamine . Where the calmodulin is from Xenopus, this corresponds to one or more of the conserved glutamic acid residues at positions E31, E67 and E140. Alternatively, the substitution may be with lysine.

Most preferably, the fluorescent protein has amino acid sequence (b) modified by substitution of the amino acid at position 148 and amino acid sequence (e) modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first EF hand of calmodulin. Where amino acid sequence (e) is Xenopus calmodulin, this corresponds to a substitution at position E31.

It is further preferred that the fluorescent protein has amino acid sequence (b) modified by substitution of the amino acid at position 148 with threonine and amino acid sequence (e) modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first EF hand of calmodulin. Where amino acid sequence (e) is Xenopus calmodulin, this corresponds to a substitution at position E31 with glutamine.

The linker sequence (c) is preferably the sequence Val-

Asp-Gly-Gly-Ser-Gly-Gly-Thr-Gly . However, the linker sequence is not limited to any particular amino acid sequence as long as the resultant protein can function as a Ca²⁺ indicator by fluorescing in a Ca²⁺-dependent manner.

Thus, the linker sequence may be any amino acid sequence that permits the fluorescent protein domains encoded by amino acid sequences (b) and (d) to interact correctly for formation of the chromophore . For example, the preferred linker sequence above may have the addition, deletion or substitution of one or more amino acids. It is preferable that the amino acid sequence comprises mainly residues with small side-chains. It is also preferable that the amino acids have hydrophilic side- chains. The linker sequence is generally between 2-20, preferably 3-10, and more preferably 5-10 amino acids in length.

A further linker sequence may be provided between amino acid sequences (d) and (e) . Such a linker sequence may be the short sequence Gly-Thr or Gly-Thr-Gly. As described above for the linker sequence (c) , a further linker sequence is not limited to any particular amino acid sequence as long as the resultant protein can function as a Ca²⁺ indicator by fluorescing in a Ca²⁺- dependent manner. Similarly, a further linker above may have the addition, deletion or substitution of one or more amino acids.

A fluorescent protein of the invention may be fused to an amino acid sequence which acts as a signal to target the fluorescent protein to a particular cell compartment. Thus, preferably, a fluorescent protein of the invention is fused to an amino acid sequence which acts as an ER retention signal. The sequence is not limited to any particular amino acid sequence as long as it can function to retain the fluorescent protein in the ER. Preferably, the amino acid sequence comprises that of a calreticulin signal, such as MLLPVPLLLGLLG AAA or MLLSVPLLLGLLGLAAAD, and a KDE motif. Further, a fluorescent protein of the invention may be fused to an amino acid sequence which acts as a sarcoplasmic reticulum targeting signal. For example, a calsequestrin signal, which is well known in the art, or an N-terminal portion thereof, may be fused to the N- terminus of the fluorescent protein.

A preferred fluorescent protein of the present invention has the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO : 6 of Fig. 4. A fluorescent protein having the amino acid sequence of one of SEQ ID NOS : 1 to 6 may have further substitution, addition and/or deletion of one or more amino acid residues, provided that the fluorescent protein retains the ability to emit fluorescence that is dependent on Ca²⁺ concentration.

A fluorescent protein of the present invention may be obtained by standard methods known in the art and is not limited to a particular method. For example, the protein may be obtained by chemical synthesis. Preferably, a protein is a recombinant protein obtained by standard recombinant techniques .

In a second aspect, the present invention provides a nucleic acid encoding a fluorescent protein of the invention.

Nucleic acid includes both DNA and RNA.

A preferred nucleic acid encoding a fluorescent protein of the invention has the nucleotide sequence shown in any one of SEQ ID NO : 7 to SEQ ID NO: 12 of Fig. 4. A nucleic acid having one of these nucleotide sequences may have substitution, addition and/or deletion of one or more nucleotides, provided that the nucleic acid encodes a fluorescent protein of the invention having the ability to emit fluorescence that is dependent on Ca²⁺ concentration .

A nucleic acid, or fragments thereof, of the present invention may be obtained by standard methods known in the art. For example, a nucleic acid may be generated by chemical synthesis or by polymerase chain reaction (PCR) using specific primers. The generation of a nucleic acid of the present invention is described in Example 1.

Methods of introducing a desired mutation into a particular nucleotide sequence are known in the art. For example, it is possible to generate a nucleic acid having a mutation by site-directed mutagenesis or by PCR using suitably designed specific oligonucleotide primers or degenerate primers. Such known techniques, in addition to recombinant techniques in general, are described in, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989.

In a further aspect, the present invention provides an expression vector comprising a nucleic acid of the invention.

The type of vector used is not limited to any particular type. For example, the vector may be one which replicates autonomously in a host cell, for example a plasmid, or one which is integrated into the host genome and is replicated along with the genome. The term "expression vector" is defined as a vector having elements necessary for transcription of a nucleic acid which are operably linked to that nucleic acid, for example, a nucleic acid of the invention. Nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. A promoter is operably linked to a nucleic acid if it effects transcription of that nucleic acid in a host cell. Such elements include a promoter, terminator and polyadenylation signal.

Suitable promoters for expression in bacterial, Drosophila or mammalian host cells are well known in the art. For example, suitable promoters for use in bacterial cells include the lac, trp and tac promoters of E. coli . For expression in Drosophila and mammalian host cells a metallothionein promoter and a viral CMV promoter may be used respectively. Suitable polyadenylation signals include a SV40 late polyadenylation signal and a BGH polyadenylation signal for Drosophila and mammalian cells respectively.

The expression vector may also contain a selectable marker. Suitable selectable markers are well known in the art, for example, antibiotic resistance genes. A neomycin resistance gene is generally preferred for use in most mammalian expression vectors.

In still a further aspect, the present invention provides a host cell transformed with a nucleic acid or an expression vector of the invention.

A transformant of the present invention may be produced by introducing a nucleic acid or expression vector of the present invention into a suitable host cell using techniques known in the art. A suitable host cell may be any cell which can express the introduced nucleic acid or expression vector. Suitable host cells including bacteria, yeast, fungi and higher eukaryotic cells are well known in the art. For example, suitable higher eukaryotic cells include Drosophila S2 cells and mammalian HeLa, CHO or 3T3 cells.

In particular, a fluorescent protein of the invention may be transformed into a Drosophila principal cell of the Malpighian tubule. Alternatively, a mammalian neuroblastoma cell may be used as a host cell. Preferably, the host cell is a CHO or 3T3 cell. Various methods of transforming suitable host cells are well known to the person skilled in the art and are described in, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989.

In yet a further aspect, the invention provides a method of measuring Ca²⁺ levels in a host cell of the invention. Preferably, the Ca²⁺ level is measured in the ER. Alternatively, the Ca²⁺ level is measured in the SR. The Ca²⁺ level is determined by measuring the fluorescence emitted by the fluorescent protein expressed by the host cell, which is dependent on the Ca²⁺ concentration. The host cell may be irradiated with light at a wavelength corresponding to the excitation wavelength of the fluorescent protein expressed by the cell. The emission spectrum may be measured or preferably the fluorescence at the emission wavelength of the fluorescent protein is measured. In another aspect, the present invention provides a method of using a fluorescent protein of the invention in a cell-based assay for detecting Ca²⁺ levels. Preferably, the assay is for detecting Ca²⁺ levels in the ER. Preferably, the cell -based assay uses mammalian cells, for example 3T3 or CHO cells. The mammalian cells may be transiently transfected or stably transformed to express a fluorescent protein of the invention. Preferably, the cells are stably transformed to benefit from the generation of a greater fluorescent signal for measurement. The cells may additionally be transfected or transformed with a further expression vector, for example one expressing a receptor for a specific ligand. Such a receptor is preferably for a calcium-inositol triphosphate (IP₃) pathway. Determination of Ca²⁺ levels may be performed according to the method of measuring Ca²⁺ levels in a host cell described above.

The cell-based assay may be one for screening drugs that work through calcium-inositol triphosphate (IP₃) pathways.

Preferably, the cell-based assay uses mammalian cells transfected or transformed to stably express a fluorescent protein of the invention. A preferred fluorescent protein of the invention for use in the cell- based assay comprises the substitution D148T/E31Q (for example, the fluorescent protein variant named "ERpicam") . The mammalian cells may be NIH 3T3 cells (American Type Culture Collection) or CHO cells as are well known in the art. Preferably, the cell-based assay provides real-time monitoring of Ca²⁺ levels in cells.

Preferably, the cell-based assay uses a multiple-well assay plate format, for example, a 96-well plate format, suitable for a high-throughput screening assay. Preferably, the multiple-well plate format assay is used in an assay to screen test compounds, for example known drugs or novel compounds, for their effects on Ca²⁺ levels in cells. Preferably, the drugs or compounds act directly or indirectly through calcium/inositol triphosphate (IP₃) pathways. In a preferred method of screening for the effect of a compound on Ca²⁺ levels and/or movement within a cell, test compounds are added to wells containing cells transfected or transformed with a fluorescent protein of the invention. Preferably, test compounds are added during real-time measurement of ER Ca⁺ levels in the cells to indicate the effect in realtime of the test compound on cell Ca²⁺ levels.

Most preferably, a cell -based assay uses mammalian cells, preferably CHO cells or 3T3 cells, expressing the fluorescent protein variant comprising the substitution D148T/E31Q (for example, "ERpicam" ) in a multiple-well assay plate, high-throughput format for screening compounds in real-time for an effect on cellular Ca²⁺ levels .

Thus, the invention provides a method of screening a test compound for an effect on Ca²⁺ levels in a host cell of the invention expressing a fluorescent protein of the invention. The method may comprise adding the test compound to the cell and detecting the fluorescence emitted by the fluorescent protein expressed in said cell. The host cell is preferably a mammalian cell. Suitable mammalian cells are known in the art and described elsewhere herein, including for example CHO cells and a 3T3 cells. The fluorescent protein of the invention expressed by the host cell is preferably one comprising the substitution D148T/E31Q (for example, "ERpicam") . Preferably, the screening is carried out in a multiple-well assay plate, high-throughput format. Preferably, the Ca²⁺ level is measured in the ER, for example, where the fluorescent protein is ERpicam having an ER retention signal . The Ca²⁺ level is determined by measuring the fluorescence emitted by the fluorescent protein expressed by the host cell, which is dependent on the Ca²⁺ concentration. The host cell may be irradiated with light at a wavelength corresponding to the excitation wavelength of the fluorescent protein expressed by the cell . The emission spectrum may be measured or preferably the fluorescence at the emission wavelength of the fluorescent protein is measured. Preferably, the screening method provides real-time monitoring of the effect of a test compound on Ca²⁺ levels in cells.

Alternatively, a fluorescent protein of the invention may be targeted to the sarcoplasmic reticulum (SR) of a host cell and used in a cell-based assay and a screening method for testing drugs, for example, for use in treating cardiac disease. The fluorescent protein may comprise the substitution D148T/E31Q, along with an SR targeting signal. Preferably, the Ca²⁺ level is measured in the SR. Suitable mammalian host cells include any cells which have SR as are well known in the art, for example, cardiac cells and muscle cells, including those derived from primary cell culture. Preferably, the host cell is a cardiac cell for cell-based assays for screening test compounds for use in cardiac disease. Brief description of the drawings

Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 shows a schematic representation of the development of fluorescent proteins according to the present invention Fig. 2 shows in vi tro properties of three embodiments of a fluorescent protein according to the present invention Fig. 3 shows real-time monitoring of ER calcium in single cells Fig. 4A to 41 shows the amino acid sequences (SEQ ID NOS: 1 to 6) and nucleotide sequences (SEQ ID NOS: 7 to 12) of embodiments of the invention Fig. 5 shows mobilisation of ER calcium by H₂0₂ in NIH 3T3 cells stably transfected with ERpicam plasmid

Detailed description of the invention

Fig. 1 shows a schematic representation of the development of three embodiments of the fluorescent protein of the present invention from the starting point of ratiometric-pericam (Fig. 1(1)). Firstly, the glutamine residue at the 12^th residue of the third EF hand of calmodulin was reverted back to a glutamic acid residue as found in wild type calmudulin. Thus, the E104Q mutation of ratiometric-pericam was reverted back in a Q104E mutation. Then, each of the conserved bidentate glutamic acid residues at the 12^th residue of each of the first, second and fourth EF hands of calmodulin of ratiometric-pericam were separately mutated to glutamine residues to give three separate variants .

Thus, residues E31, E67 and E140 of the Xenopus calmodulin in ratiometric-pericam were separately substituted with glutamine residues. The E31 variant is shown in Fig. 1(2) . This was achieved by constructing plasmids containing the required mutations in the sequence according to standard known techniques and specifically as described in Example 1. Thus, plasmids were constructed for variants of ratiometric-pericam having the reversion Q104E and one of the mutations E31Q, E67Q or E140Q. This was followed by transformation of the plasmids into host cells to enable expression of the proteins as described in Example 2. Fluorescence of the proteins produced by the transformants was measured as described in Example 2.

However, the three variant proteins were all found to have lost their fluorescent properties. Without being bound by any particular theory, it is possible that the substitutions alter the interaction between calmodulin and the target peptide of calmodulin, M13 , causing an out-of-plane distortion between the two halves of EYFP.

In an attempt to restore fluorescence, the linkers in the proteins were modified in order to make them more flexible. For example, as shown in Fig. 1(3), an additional glycine residue was added to the short linker between the second of the two fluorescent protein domains and the calmodulin domain to give the linker sequence Gly-Thr-Gly. However, the modifications made to the linkers did not allow the two halves of EYFP to interact correctly for fluorophore formation and so did not restore fluorescent properties to the proteins.

Inverse-pericam is identical in structure to ratiometric- pericam except for a single amino acid substitution at position 148 in the mutant YFP domain in which aspartic acid is substituted with threonine (D148T) . This substitution alters the fluorescent properties of the protein such that fluorescence is decreased in the presence of Ca²⁺. In a further attempt to restore fluorescence to the three variants, the aspartic acid at position 148 was similarly substituted for threonine as shown in Fig. 1(4). Plasmids were constructed as described above, and contained mutations to effect the D148T substitution in combination with mutations for the calmodulin domain substitutions. Expression of the proteins in transformed cells and measurement of fluorescence were carried out as described in Examples 2 and 3.

Given that the D148T substitution in inverse-pericam has the effect of decreasing fluorescence, surprisingly, this substitution in combination with a substitution of any of the conserved glutamic acid residues of the calmodulin domain has the effect of restoring fluorescent properties to the non-fluorescent variants. These proteins with restored fluorescent properties, like inverse-pericam, have a single excitation wavelength.

Subsequently, each of these three variants, namely ratiometric-pericam variants having the reversion Q104E and the substitution D148T/E31Q, D148T/E67Q or D148T/E140Q, respectively, were generated with a calreticulin signal (MLLPVPLLLGLLGLAAA) and a KDEL motif to ensure their retention in the ER of a cell . This was again achieved by constructing plasmids using standard known techniques and specifically as described in Example 1. The D148T/E31Q variant was named "ERpicam". Fig. 2 shows in vi tro properties of the three variants, determined as described in Example 2. The results of Ca²⁺ and pH titrations are shown in Fig. 2. As can be seen from Fig. 2, ERpicam has a very wide sensitivity to Ca²⁺ and is very robust in terms of pH sensitivity. At pH 7.0, which is the approximate pH of the ER, ERpicam functions at near maximum efficiency (Fig. 2A) . It has a single excitation and a single emission wavelength. Although this does not allow the quantitative measurements that were possible with the original ratiometric-pericam, monitoring of Ca²⁺ changes is very simple. The calculated Ka of ERpicam is 4 μM (Fig. 2D) . Thus, the affinity of ERpicam for Ca²⁺ is less than that of any of the pericam proteins (reported K_&s of 0.7 μM, 1.7 μM and 0.2 μM for flash-, ratiometric- and inverse- pericam respectively, EP 1 238 982 A, Table 1) indicating the reduced sensitivity of ERpicam to Ca⁺.

Thus, these results indicate that ERpicam has the required properties to make it suitable for use as an indicator of Ca²⁺ levels in the ER. Provided with a SR targeting signal instead of an ER retention signal, the D148T/E31Q variant is also suitable for use as an indicator of Ca⁺ levels in the SR.

The other two variants, D148T/E140Q and D148T/E67Q, have calculated K^s of 0.5 μM and 0.05 μM, respectively (Fig. 2C,F and Fig. 2B,E) . Similarly, these variants have the required properties to be suitable for use as indicators of Ca²⁺ levels in the ER or the SR.

Fig. 3 shows the results of real-time monitoring of ER

Ca²⁺ levels in single cells. The ERpicam construct was used to transform Drosophila and mammalian cell lines to allow expression of ERpicam as described in Example 3. In vivo fluorescence measurements were carried out as described in Example 3. In both cell lines the fluorescent protein produced a bright signal. Fig. 3A shows Ca²⁺ levels in the ER of a single principal cell of the Drosophila Malpighian tubule. The neuropeptide capa- 1 is known to stimulate an IP₃-induced cytoplasmic calcium increase (Kean et al . (2002) Am J Physiol Regul Integr Comp Physiol 282 R1297-307; Pollock et al . (2003) J Exp Biol 206, 901-911) . Thus, Fig. 3A demonstrates the existence of a non-ER-based IP₃-releasable Ca²⁺ pool in renal epithelium of Drosophila . Fig. 3B shows Ca²⁺ levels in the ER of a single mammalian neuroblastoma cell starved of serum. Addition of serum stimulates the cell, causing the depletion of ER Ca²⁺ stores.

The fluorescent proteins can be used in cell-based assays for detecting Ca²⁺ levels in the ER. Mammalian host cells can be used in assays for screening drugs that work through calcium-inositol triphosphate (IP₃) pathways. An assay plate with wells containing mammalian cells transfected or transformed with the ERpicam construct is placed in a platereader (for example, Berthold or Genetix) . A single excitation wavelength of 485 nm is used to excite the cells by irradiating them with UV light filtered through a 485 nm filter. Emission at a single wavelength of 525 nm is detected through a 525 nm filter. A compound to be assayed, for example a drug, is injected in real-time into the plate wells containing the cells and the change in fluorescence is measured in realtime. Unlike aequorin-based measurement of ER Ca²⁺ levels, the cells do not require treatment or preparation prior to the assay. Thus, the fluorescent proteins allow simple cell-based assays to be performed requiring measurement of only single excitation and emission wavelengths .

Furthermore, the effect of a drug on a specific receptor may be tested by using host cells that have additionally been transfected or transformed with the coding sequence for that receptor. The assay described above is carried out to determine whether or not there is any interaction between the test drug and the expressed receptor.

ERpicam expression has been demonstrated in the ER of mammalian cells in cell-based assays as described in Example 4. NIH 3T3 cells were stably transfected with ERpicam (ERpicam NIH 3T3 cells) . Confocal images (not shown) were obtained which show fluorescence in groups of ERpicam NIH 3T3 cells. Fluorescence was associated with intracellular compartments of the cells, but not the nuclei . Further confocal images (not shown) were obtained of ERpicam NIH 3T3 cells in which the ER had been specifically stained with an ER tracker dye. Low magnification images demonstrate that ERpica -associated fluorescence and staining by the ER tracker co-localize in the cytoplasm of the cells. High magnification images demonstrate ERpicam-associated fluorescence in the ER; there is precise co-localization of the ER tracker with the ERpicam fluorescence signal.

ERpicam has been used for monitoring fluorescence changes induced by calcium mobilising agents in cell-based assays as described in Example 4. Figure 5 shows the results of mobilization of ER Ca²⁺ by H₂0₂ in ERpicam NIH 3T3 cells as shown by fluorescence intensity in the ER over time. Figure 5 is a typical real-time trace (in seconds) of Ca²⁺ mobilization from ER in control PBS-treated ERpicam NIH 3T3 cells (lower trace) ; and in H₂0₂-treated cells (25 μM final concentration H₂0₂, injected at t=5s (arrow) via injectors into wells) (upper trace) . An increase in fluorescence intensity was observed when the H₂0₂ was added to the cells, indicating a decrease in ER Ca²⁺ levels due to mobilization of Ca²⁺ from the ER (ERpicam has the D148T substitution of inverse pericam giving the fluorescent property of decreasing fluorescence as Ca²⁺ levels increase) .

Examples

Example 1. Plasmid construction

Ratiometric-pericam and inverse-pericam were amplified and cloned into the pCRT7/NT TOPO bacterial expression vector (Invitrogen) . Required mutations were introduced using PCR mutagenesis with Pfu polymerase (Stratagene) . ERpicam was assembled by fusion PCR of the PCR fragments of the calreticulin signal (pSVAQERK construct, Molecular Probes) and the Q104E, D148T/E31Q variant of ratiometric- pericam. The primers included the KDEL motif and also EcoRI and Notl restriction sites. The fusion PCR was cloned into pcDNA3.1 TOPO (Invitrogen) to generate pcDNA3.1-ERpicam, and also directly cut and cloned into the P-element vector pUAST.

Example 2. Bacterial expression and in vi tro fluorescence measurements pCRT7/NT vectors containing the mutated ratiometric- pericam variants, and which incorporate a N-terminal HIS- tag, were transformed into BL21 ROSETTA (Novagen) .

Cultures were seeded and grown at 37°C for 3 h, induced with ImM IPTG and left to express for 20 h at RT. Cells were spun down, resuspended in IX binding buffer (Novagen) containing protease inhibitors and lysed using sonication (Vibra-Cell) . The lysate was spun down and the soluble fraction was purified using a His-bind column purification kit (Novagen) .

The Ca²⁺ titrations were carried out using Ca²⁺ buffered solutions (Molecular Probes) and pH titrations in 10 mM Na borate, 10 mM K phthalate, lOmM HEPES, 125mM KC1 , 20 mM NaCl, 0.5 mM MgCl₂ and either 10 mM CaCl₂ or 3 mM EGTA. These titrations were performed in a Berthold Mithras platereader, with samples excited at 485 nm and read at 525 nm. The excitation spectra was analysed on a custom built Cairn spectrophotometer .

Example 3. Expression in Drosophila and mammalian cell lines and in vivo fluorescence measurements Transgenic Drosophila were generated using standard procedures of germ line transformation (Spradling A C and Rubin G M(1982) Science 218, 341-347) and utilising the UAS/GAL4 system (Brand A H and Perrimon N (1993)

Development 118, 401-415). Thus, transgenic flies were generated carrying a fluorescent protein sequence under the control of the UAS promoter. These flies were then crossed with specific GAL4 enhancer trap fly lines. The resulting offspring expressed the fluorescent protein in the principal cells of the Malpighian tubules. Malpighian tubules were dissected from the resultant adult transgenic flies and carefully stuck, in PBS, to the bottom of a glass-bottomed dish (Mattek) that had been treated with poly-L-lysine . The PBS was immediately removed and 3 ml of Schneider's solution was added. The samples were left for at least an hour before imaging to allow the tubules to recover from being in PBS and to prevent the interference of any stimuli that may have occurred within the fly prior to dissection.

Mammalian neuroblastoma cells were transiently transfected with the pcDNA3.1-ERpicam construct in a sterile glass-bottomed dish (Mattek or Iwaki) according to standard procedures. Alternatively, stable mammalian cell lines expressing the fluorescent protein could be transformed according to standard procedures . Individual cells were then imaged.

For both Drosophila Malpighian tubules and mammalian neuroblastoma cells, imaging was performed using a Zeiss 510 Meta confocal system coupled to an inverted Zeiss microscope. The fluorescent proteins were excited with an Argon 488 laser and the emission filtered through a 505-530 band pass filter. A 20x objective was used for all live imaging of Drosophila Malpighian tubules and a 63x objective was used for the mammalian neuroblastoma cells. The real-time images of the cells of the tubules or neuroblastoma cells expressing ERpicam were captured and average fluorescence values for specified regions of interest (ROI) were calculated. These values were subtracted from an arbitrary higher value to give relative values of [Ca²⁺] _ER content .

Example 4. Expression in cell-based assays

Cell lines

NIH 3T3 cells (American Type Culture Collection) were used to study the expression of a variant fluorescent protein of the invention in the ER of mammalian cells.

Cells were cultured in 75 cm² cell culture flasks with

Dulbecco's Modified Eagle's Medium (DMEM) , 10% (v/v)

Newborn Calf Serum and 1% (v/v) penicillin/streptomycin (all Invitrogen) . Cells were passaged with 5ml of trypsin/EDTA solution (Sigma) , re-suspended and plated at 5xl0⁵ cells/well in 6-well cell culture plate without antibiotics. Cultures were maintained at 37°C, 5% C0₂ overnight. The next day, medium was removed and replaced with DMEM only for 1 hour. Cells were then transfected with Lipofectin reagent (Invitrogen) following manufacturer's instructions using 10 μl Lipofectin reagent and 4 μg of Endo-free purified (Quiagen) ERpicam plasmid (comprising the Q104E and D148T/E31Q mutations, a calreticulin signal and a KDEL motif, as described above) . After 24 h the medium was removed and replaced with DMEM and 10% (v/v) NCS and cultures maintained for a further 24 h. The next day the medium was removed, cells trypsinised and re-plated in 75 cm² flasks with DMEM, 10% NCS, 400 μg/ml Geneticin (Invitrogen) and incubated overnight. Cells were passaged every 3-4 days in Geneticin-containing medium at 90% confluency until derivation of a stable ERpicam cell line (ERpicam NIH 3T3 cells) .

I_. Cell-based assays showing ERpicam expression in the ER of mammalian cells

ERpicam NIH 3T3 cells were cultured on coverslips inside a Petri dish filled with DMEM, 10% v/v NCS and 400 μg/ml Geneticin (Invitrogen) and incubated at 37° C, 5 % C0₂ overnight. The next day, the medium was removed and pre- warmed (37 °C) medium (DMEM, 10% v/v NCS) added. Fluorescence was viewed with a Zeiss Axiocam imaging system using appropriate excitation and emission filters for GFP (488/505-535 nm, respectively) . The confocal images obtained (not shown) showed fluorescence in groups of ERpicam NIH 3T3 cells, indicating ERpicam expression.

The fluorescence was associated with intracellular compartments of the cells, indicating the presence of ERpicam in the ER. The cells were also treated with DAPI (4', 6 ' -diamidino-2-phenylindole hydrochloride) using techniques known in the art, to localize nuclei. A clear lack of fluorescence was observed in cell nuclei, indicating the specificity of targeting of ERpicam to the ER.

Specific staining of ER in ERpicam NIH 3T3 cells was achieved using an ER tracker dye. This ER-Tracker Blue- White DPX dye (Molecular Probes, E-12353) is a photo- stable probe that is selective for the endoplasmic reticulum (ER) in live cells. Culture and preparation of ERpicam NIH 3T3 cells for ER imaging was performed in a similar manner to that for imaging ERpicam fluorescence as described above, except that cells were incubated with pre-warmed (37°C) DMEM, 10% v/v NCS medium containing 500 nM ER tracker dye for 30 minutes under growth conditions (37°C, 5% C0₂) . The loading solution was then replaced with fresh growth medium and staining observed using a

Zeiss Axiocam imaging system at an excitation wavelength of -374 nm. The confocal images obtained (not shown) showed co-localization of ERpicam with the ER in ERpicam NIH 3T3 cells expressing ERpicam counterstained with the ER tracker dye. Fluorescence (visualized as green) in ERpicam NIH 3T3 cells indicated ERpicam expression. The ER tracker dye (visualized as red) localized the ER in the cells. The two images merged showed precise co- localization of the two signals (visualized as yellow) . Low magnification images showed that ERpicam-associated fluorescence and staining by ER tracker co-localised in the cytoplasm of ERpicam NIH 3T3 cells. High magnification images showed ERpicam-associated fluorescence in the ER and the precise co-localisation of ER tracker dye signal with ERpicam fluorescence signal .

II . Cell -based assays showing use of ERpicam in monitoring fluorescence changes induced by calcium mobilising agents

ERpicam NIH 3T3 cells were trypsinised, re-suspended in serum-free DMEM and plated in 96-well plates at 10^s cells/well. After 3 hours, basal and stimulated fluorescence intensities were measured using a Berthold Mithras platereader as previously described, using filters at the following wavelengths: Excitation (485 nm) and emission (535 nm) . Basal fluorescence indicating basal ER Ca²⁺ levels was measured in control PBS-treated ERpicam NIH 3T3 cells. H₂0₂ was used to stimulate mobilization of Ca²⁺ from the ER of cells (Kamsler A and Segal M (2003) J Neurosci 23, 269-276) . Stimulated fluorescence indicating Ca²⁺ mobilization from the ER was measured in H₂0₂-treated cells (25 μM final concentration H₂0₂, injected at t=5s via injectors into wells) . An increase in fluorescence intensity was observed when the H₂0₂ was added to the cells, indicating mobilization of Ca²⁺ from the ER and a resulting decrease in ER Ca²⁺ levels .

Conclusions

ERpicam is expressed only in the ER of ERpicam NIH 3T3 cells. This, together with the other data described above from in vi tro measurements and from in vivo measurements in Drosophila and mammalian cell lines, indicates that ERpicam can be correctly targeted to the

ER in other cell types, and demonstrates the applicability of ERpicam for use in any cell type of choice . Screening of ER calcium activity using ERpicam has been demonstrated in a mammalian cell line in a 96-well format assay using a standard platereader. The ability to determine the effect of a compound on Ca²⁺ levels in mammalian cells in real-time has been demonstrated using this assay format, with the assay compound added to the cells and any resulting change in fluorescence indicating change in ER Ca²⁺ levels measured in real-time. This demonstrates the possible applications for ERpicam, for example, in high-throughput drug screening in mammalian cells .

References The references cited herein are all expressly incorporated by reference .

Baird G S, Zacharias D A, Tsien R Y (1999) . Circular permutation and receptor insertion within green fluorescent proteins. Proc Nat Acad Sci USA 96, 11241- 11246

Brand A H and Perrimon N (1993) . Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415

Kean L, Cazenave W, Costes L, Broderick K E, Graham S, Pollock V P, Veenstra J A, Davies S A, Dow J A (2002) . Two nitridergic peptides are encoded by the gene capabili ty in Drosphila melanogaster. Am J Physiol Regul Integr Comp Physiol 282, R1297-307

MacPherson M R, Pollock V P, Broderick K E, Kean L,

O'Connell F C, Dow J A, Davies S A (2001) . Model organisms: New insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosphila melanogaster . Am J Physiol Cell Physiol 280, C394-C407

Miyawaki A, Llopis J, Heim R, McCaffery J M, Adams J A, Ikura M, Tsien R Y (1997) . Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature 388, 882-887

Miyawaki et al . (1999). Proc. Natl . Acad. Sci. USA 96, 2135-2140

Nagai T, Sawano A, Park E S, Miyawaki A (2001) . Circularly permuted fluorescent proteins engineered to sense Ca²⁺. Proc Nat Acad Sci USA 98, 3197-3202

Ormδ et al . (1996). Science 273, 1392-1395

Osawa M, Tokumitsu H, Swindells M B, Kurihara H, Orita M, Shibanuma T, Furuya T, Ikura M (1999) . A novel target recognition revealed by calmodulin in complex with Ca²⁺- calmodulin-dependent kinase kinase. Nat Struct Biol 6(9), 819-824

Pollock V P, Radford J C, Pyne S, Hasan G, Dow J A, Davies S A (2003) . norpA and itpr mutants reveal roles for phospholipase C and inositol (1 , 4 , 5) -triphosphate receptor in Drosphila melanogaster renal function. J Exp Biol 206, 901-911

Rosay P, Davies S A, Yu Y, Sozen A, Kaiser K, Dow J A (1997) . Cell-type specific calcium signalling in a

Drosphila epithelium. J Cell Sci 110, 1683-1692 Spradling A C and Rubin G M (1982) . Transposition of cloned P elements into Drosophila germ line chromosomes Science 218, 341-347

Terhzaz S, O'Connell F C, Pollock V P, Kean L, Davies S A, Veenstra J A, Dow J A (1999) . Isolation and characterization of a leucokinin-like peptide of Drosphila melanogaster. J ExpBiol 202, 3667-3676

Tsin et al . (1998) . Ann. Rev. Biochem. 67, 509-544

Claims

Claims

1. A fluorescent protein comprising, in the direction from the N terminus to the C terminus, (a) an amino acid sequence of a target peptide of calmodulin; (b) an amino acid sequence from the n^th amino acid from the N terminus to the C terminus of a fluorescent protein where n represents an integer of 140 to 148, wherein the amino acid sequence is modified by substitution of the amino acid at position 148; (c) a linker sequence of 2 to 20 amino acids; (d) an amino acid sequence from the first amino acid to the (n-l)^th amino acid from the N terminus of the fluorescent protein in (b) ; (e) an amino acid sequence of calmodulin, wherein the amino acid sequence is modified by substitution of one or more of the conserved bidentate glutamic acid residues at the 12^th residue of the first, second or fourth EF hand of calmodulin; and wherein the fluorescent protein can emit fluorescence that is dependent on Ca²⁺ concentration.

2. The fluorescent protein according to claim 1, wherein amino acid sequence (b) is that of a yellow fluorescent protein mutant having the substitutions V68L, Q69K and Y203F.

3. The fluorescent protein according to claim 1 or claim 2, wherein in amino acid sequences (b) and (d) , n is 145.

4. The fluorescent protein according to any one of claims 1 to 3 , wherein amino acid sequence (b) is modified by substitution of the amino acid at position 148 with threonine.

5. The fluorescent protein according to any one of the preceding claims, wherein amino acid sequence (e) is modified by substitution of one or more of the conserved glutamic acid residues at the 12^th residue of the first, second or fourth EF hand of calmodulin with glutamine or lysine.

6. The fluorescent protein according to any one of the preceding claims, wherein amino acid sequence (e) is modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first or fourth EF hand of calmodulin.

7. The fluorescent protein according to any one of the preceding claims, wherein amino acid sequence (b) is modified by substitution of the amino acid at position 148 and amino acid sequence (e) is modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first EF hand of calmodulin.

8. The fluorescent protein according to claim 7, wherein amino acid sequence (b) is modified by substitution of the amino acid at position 148 with threonine and amino acid sequence (e) is modified by substitution of the conserved glutamic acid residue at the 12^th residue of the first EF hand of calmodulin with glutamine .

9. The fluorescent protein according to any one of the preceding claims, wherein the linker sequence (c) is Val-

Asp-Gly-Gly-Ser-Gly-Gly-Thr-Gly.

10. The fluorescent protein according to any one of the preceding claims, wherein the fluorescent protein has the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6.

11. The fluorescent protein according to any one of the preceding claims further comprising a linker sequence between amino acid sequence (d) and amino acid sequence

(e) .

12. The fluorescent protein according to claim 11, wherein the further linker sequence is Gly-Thr or Gly- Thr-Gly.

13. The fluorescent protein according to any one of the preceding claims, wherein the target peptide of calmodulin is M13.

14. The fluorescent protein according to any one of the preceding claims, wherein the protein is fused to an amino acid sequence which acts as an endoplasmic reticulum retention signal.

15. The fluorescent protein according to claim 14, wherein the amino acid sequence comprises that of a calreticulin signal and a KDEL motif.

16. The fluorescent protein according to any one of claims 1 to 13, wherein the protein is fused to an amino acid sequence which acts as a sarcoplasmic reticulum targeting signal.

17. The fluorescent protein according to claim 16, wherein the amino acid sequence comprises that of a calsequestrin signal .

18. A nucleic acid encoding the fluorescent protein of any one of the preceding claims .

19. The nucleic acid according to claim 18 wherein the nucleic acid has the nucleotide sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12.

20. An expression vector comprising the nucleic acid of claim 18 or claim 19.

21. A host cell transformed with the nucleic acid of claim 18 or claim 19 or the expression vector of claim 20.

22. The host cell according to claim 21, further transformed with a nucleic acid or expression vector encoding a receptor for a calcium-inositol triphosphate (IP₃) pathway.

23. The host cell according to claim 21 or claim 22, which is a mammalian cell .

24. A method of measuring Ca²⁺ concentration in the host cell of any one of claims 21 to 23 by detecting the fluorescence emitted by the fluorescent protein expressed in said cell.

25. The method according to claim 24, wherein the Ca⁺ concentration is measured in the endoplasmic reticulum of the host cell .

26. Use of the fluorescent protein of any one of claims 1 to 17 in a cell-based assay for detecting Ca²⁺ levels.

27. The use according to claim 26, wherein the Ca²⁺ levels are detected in the endoplasmic reticulum of the cell .

28. The use according to claim 26 or 27, wherein the cell is a mammalian cell.

29. A method of screening a test compound for an effect on Ca²⁺ levels in a cell of any one of claims 21 to 23, comprising adding the test compound to the cell and detecting the fluorescence emitted by the fluorescent protein expressed in said cell.