AU2002326047A1 - Fluorescent proteins - Google Patents

Description

FLUORESCENT PROTEINS

The present invention relates to novel blue-shifted variants of the fluorescent protein GFP (Green Fluorescent Protein) having improved fluorescence properties.

The use of fluorescent proteins derived from Aequorea victoria has revolutionised research into many cellular and molecular-biological processes. For example, use of green fluorescent protein (GFP) allows researchers to label proteins within cells with an intrinsic fluor, so obviating the requirement to perform chemical labelling of proteins, and allowing development of assays of biological processes in intact living cells.

US 5491 084 describes the use of GFP as a biological reporter. Early applications of GFP as a biological reporter (Chalfie et al. Science, (1 994),

263, 802-5; Chalfie, et al, Photochem.Photobiol., (1 995), 62(4), 651 -6) used wild type (native) GFP (wtGFP), but these studies quickly demonstrated two areas of deficiency of wtGFP as a reporter for use in mammalian cells. The protein being derived from a poikilothermic marine organism does not undergo protein folding efficiently when expressed in mammalian cells cultured at 37 °C, resulting in weak fluorescence.

Consequently, significant effort has been expended to produce variant mutated forms of GFP with properties more suitable for use as an intracellular reporter.

A number of mutated forms of GFP with altered spectral properties have been described. A variant-GFP (Heim et al. (1 994) Proc.Natl.Acad.Sci. 91 , 1 2501 ) contains a Y66H mutation which blue-shifts the excitation and emission spectrum of the protein. However, this protein is only weakly fluorescent and rapidly photo-bleaches following excitation. WO96/27675 describes two variant GFPs, obtained by random mutagenesis and subsequent selection for brightness, which contain the mutations V163A and V163A + S1 75G, respectively. These variants were shown to produce more efficient expression in plant cells relative to wtGFP and to increase the thermotolerance of protein folding. The double mutant V1 63A + S1 75G was observed to be brighter than the variant containing the single V1 63A mutant alone. This mutant exhibits a blue-shifted excitation peak.

US 61 721 88 describes variant GFPs wherein the amino acid in position 1 preceding the chromophore has been mutated to provide an increase of fluorescence intensity. Such mutations include F64I, F64V, F64A, F64G and F64L, with F64L being the preferred mutation. These mutants result in a substantial increase in the intensity of fluorescence of GFP without shifting the excitation and emission maxima. F64L-GFP has been shown to yield an approximate 6-fold increase in fluorescence at 37 °C due to shorter chromophore maturation time.

In addition to the single mutants or randomly derived combinations of mutations described above, a variety of variant-GFPs have been created which contain two or more mutations deliberately selected from those described above and other mutations, and which seek to combine the advantageous properties of the individual mutations to produce a protein with expression and spectral properties which are suited to use as a sensitive biological reporter in mammalian cells.

US 61 94548 discloses GFPs with improved fluorescence and folding characteristics at 37 °C that contain, at least, the changes F64L and V1 63A and S1 75G.

US 5777079 describes a blue fluorescent protein (BFP) containing F64L, S65T, Y66H and Y145F mutations. This is referred to as BFP (Blue Fluorescent Protein), because it emits blue fluorescence by UV excitation (R. Heim et al. Curr. Biol. (1 996), 6, 1 78-182; R. Heim et al. Proc. Natl. Acad. Sci. USA, (1 994), 91, 1 2501 -1 2504). However, this BFP was very dim and it experienced severe photo-bleaching as compared to green fluorescent protein. US 61 94548 describes a further BFP containing the F64L, Y66H, Y145F and L236R substitutions. This patent also discloses a mutant containing: F64L, Y66H, Y145F, V1 63A, S1 75G, and L236R. Further mutants are described comprising the Y66H, Y145F, V1 63A and S1 75G mutations; and the F64L, Y66H, and Y145F mutations. Further optional mutations are described at S65T and Y231 L. These mutants are more photostable than those described in US 5777079, but they still suffer from some photo-bleaching effects.

The present invention provides novel engineered derivatives of blue fluorescent protein (BFP) and nucleic acids that encode engineered BFPs which exhibit more stable fluorescence properties and have different excitation spectra and/or emission spectra relative to wtGFP when expressed in non-homologous cells at temperatures above 30 °C, and when excited at about 390 nm. In particular, the invention provides novel fluorescent proteins that fluoresce in the blue region of the spectrum ("BFPs").

Moreover, it has surprisingly been found that the novel engineered derivatives of blue fluorescent protein have a cellular fluorescence that is more stable than that of BFPs previously described. The invention also relates to compositions such as expression vectors and cells that comprise either the mutant nucleic acids or the mutant protein gene products. The mutant fluorescent proteins produced using the method of the invention provide a means for detecting multiple GFP reporters in mammalian cells at lower levels of expression and/or increased sensitivity relative to wtGFP. This greatly improves the usefulness of fluorescent proteins in studying cellular functions in living cells. ln a first aspect of the invention, there is provided a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S1 75; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP.

Suitably, the amino acid F at position 64 may be substituted by an amino acid selected from the group consisting of L, I, V, A and G, thereby providing F64L, F64I, F64V, F64A, or F64G substitutions. Preferably, the amino acid substitution at position 64 is the F64L substitution.

Suitably, the amino acid Y at position 66 may be substituted by an amino acid selected from H and F, thereby providing Y66H or Y66F substitutions. Preferably, the amino acid substitution at position 66 is the Y66H substitution.

Suitably, the amino acid S at position 1 75 may be substituted by an amino acid selected from the group consisting of G, A, L, I and T, thereby providing S1 75G, S1 75A, S1 75L, S1 75I and S1 75T substitutions. Preferably, the amino acid substitution at position 1 75 is the S175G substitution.

Optionally, the fluorescent protein according to the first aspect may be further modified by amino acid substitution selected from one or more of the following: S72A, Y1 45F, N 146I, M 1 53T, N1 98S and T203I. Suitably, the novel fluorescent proteins exhibit high fluorescence in cells expressing them when said cells are incubated at a temperature of 30 °C or above, preferably at a temperature of from 32 °C to 39 °C, more preferably from 35 °C to 38 °C and most preferably at a temperature of about 37 °C.

Preferably, the fluorescent protein according to the first aspect has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein having the sequence: SEQ ID No.2.

A preferred protein according to the present invention is a protein in which, in relation to SEQ ID No.2 of GFP, the amino acid F at position 64 has been substituted by L, the amino acid Y at position 66 has been substituted by H and the amino acid S at position 1 75 has been substituted by G, and is shown herein as having the amino acid sequence as set forth in SEQ ID No.3.

Suitably, the GFP or functional GFP-analogue used to derive the fluorescent protein may be obtained from any convenient source. For example, native GFP derived from species of the genus Aequorea, suitably Aequorea victoria. The chromophore in wtGFP from Aequorea victoria is at positions 65-67 of the predicted primary amino acid sequence (SEQ ID No.2). In a preferred embodiment, the GFP is derived from Aequorea victoria.

The modified proteins of the present invention may be produced by introducing mutations in a sequence of the nucleic acid that encodes the protein. As used herein, a preferred sequence of the gene encoding wtGFP is derived from Aequorea victoria, published by Chalfie et al, (Science, (1 994), 263, 802-5) disclosed as SEQ. ID. No1 (Figure 1 ). The corresponding amino acid sequence is shown in SEQ. ID. No2 (Figure 2). Alternative sequences of the GFP gene may be used, for example, the nucleotide (and predicted amino acid) sequences of the GFP gene described by Prasher et al, (Gene (1 992), 1 1 1 , 229) and the sequences as disclosed in WO 97/1 1 094. In addition, alternative gene sequences that encode the fluorescent protein may incorporate a consensus Kozak nucleotide sequence (Kozak, M., Cell (1 986), 44, 283), or preferred mammalian codons, to provide improved translation in mammalian systems. The nucleotide sequence corresponding to the fluorescent protein may also encode useful restriction enzyme sites and additional elements such as target sites for enzymes and purification tags. Methods for incorporation of a Kozak region, preferred mammalian codons, restriction enzyme sites, enzyme sites and purification tags are well known in the art and may result in the incorporation of amino acid residues and a change in numbering of amino acid residues in the fluorescent protein relative to the wtGFP numbering in the sequence provided.

Herein, the abbreviations used for the amino acids are those stated in J.Biol.Chem., (1 968), 243, 3558.

In a second aspect of the invention, there is provided a fusion compound comprising a protein of interest fused to a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S1 75; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP.

In the context of the present invention, the term "protein of interest" is intended also to encompass polypeptides and peptide fragments. Examples of such proteins of interest include: NFKB and subunits thereof, RAC1 , PLC domains, MAPKAP2, PKC, Cytochrome C, RHO, β-actin, STAT6, protein kinase C isotypes, LAMP1 /2 TGN, ATP7A TGN and GLUT4.

In a third aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S1 75; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP.

Preferably, the nucleic acid molecule according to the third aspect encodes a fluorescent protein having an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein having the sequence: SEQ ID No.2.

In a particular embodiment of the third aspect, the nucleic acid molecule comprises a nucleotide sequence encoding a fluorescent protein derived from Green Fluorescent Protein (GFP) or any functional GFP analogue according to the invention fused to a nucleotide sequence encoding a protein of interest.

Preferably, the nucleic acid molecule is a construct comprising a DNA sequence.

Preferably, the nucleic acid molecule encodes a fluorescent protein having an amino acid sequence consisting of SEQ ID No.3. As is well known, a single amino acid may be encoded by more than one nucleotide codon and thus each of the above nucleotide sequences may be modified to produce an alternative nucleotide sequence that encodes the same peptide. Thus, the preferred embodiments of the invention include alternative DNA sequences that encode the preferred proteins as previously described. It is to be understood that the preferred proteins (and the nucleic acid sequences from which they are derived), may include additional residues, particularly N- and C-terminal amino acids, or 5'- or 3'-nucleotide sequences, and still be essentially as described herein.

Suitably, the DNA construct encoding the novel fluorescent proteins may be prepared synthetically by established methods, e.g. the phosphoramidite method described by Beaucage and Caruthers, (Tetrahedron Letters (1 981 ), 22, 1 859-1 869), or the method described by Matthes et al., (EMBO Journal (1 984), 3, 801 -805). According to the phosphoramidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned into suitable vectors.

The DNA construct encoding the fluorescent protein may also be prepared by recombinant DNA methodology, for example cDNA cloning. See for example, Sambrook, J. et al (1 989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press.

The DNA construct may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4683202 or by Saiki et al (Science (1 988), 239, 487-491 ). A review of PCR methods may be found in PCR Protocols, (1 990), Academic Press, San Diego, California, USA.

The gene sequence encoding the fluorescent protein may be joined in- frame with a gene encoding the protein of interest and the desired fusion protein produced when inserted into an appropriate expression vector. For example, polymerase chain reaction or complementary oligonucleotides may be employed to engineer a polynucleotide sequence corresponding to the fluorescent protein, 5' or 3' to the gene sequence corresponding to the protein of interest. Alternatively, the same techniques may be used to engineer a polynucleotide sequence corresponding to the fluorescent protein sequence 5' or 3' to the multiple cloning site of an expression vector prior to insertion of a gene sequence encoding the protein of interest. The polynucleotide sequence corresponding to the fluorescent protein sequence may comprise additional nucleotide sequences to include cloning sites, linkers, transcription and translation initiation and/or termination signals, labelling and purification tags.

In a fourth aspect, there is provided an expression vector comprising suitable expression control sequences operably linked to a nucleic acid molecule according to the present invention. The DNA construct of the invention may be inserted into a recombinant vector, which may be any vector that may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding a fluorescent protein of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fluorescent protein of the invention.

The promoter may be any DNA sequence which shows transcriptional activity in a suitable host cell of choice, (eg. a bacterial cell, a mammalian cell, a yeast cell, or an insect cell) for expressing a fluorescent protein. The promoter may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the

DNA sequence encoding the fluorescent protein of the invention in mammalian cells are the CMV promoter (US 51 68062, US5385839), Ubiquitin C promoter (Wulff, M. et al., FEBS Lett. ( 1 990), 261 , 101 -105), SV40 promoter (Subramani et al., Mol. Cell Biol. (1 981 ), 1, 854-864) and MT-1 (metallothionein gene) promoter (Palmiter et al., Science (1 983), 222, 809-814). An example of a suitable promoter for use in insect cells is the polyhedrin promoter (US 4745051 ; Vasuvedan et al., FEBS Lett., ( 1 992) 31 1 , 7-1 1 ). Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem., ( 1 980), 255, 1 2073-1 2080; Alber and Kawasaki, J. Mol. Appl. Gen., (1 982), 1, 41 9-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1 982), or the TPI 1 (US 459931 1 ) or ADH2-4c (Russell et al., Nature, (1 983), 304, 652-654) promoters.

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amy/o/iquefaciens BAN amylase gene, the Baci/lus subti/is alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or the Escherichia coli lac, trp or tac promoters. The DNA sequence encoding the novel fluorescent proteins of the invention may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TP11 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators. The vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).

The vector may further comprise a DNA sequence enabling internal ribosomal entry and expression of two proteins from one bicistronic transcript mRNA molecule. For example, the internal ribosomal entry sequence from the encephalomyocarditis virus (Rees S, et al, BioTechniques (1996), 20, 102-1 10 and US 49371 90).

The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication.

When the host cell is a yeast cell, examples of suitable sequences enabling the vector to replicate are the yeast plasmid 2μ replication genes REP 1 -3 and origin of replication.

The vector may also comprise selectable markers, such as a gene that confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, puromycin, neomycin or hygromycin.

The procedures used to ligate the DNA sequences coding for the fluorescent protein of the invention, the promoter and optionally the terminator and/ or targeting sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (eg. Sambrook et al., op. cit.).

In a fifth aspect of the invention, there is provided a host cell transformed or transfected with a DNA construct comprising an expression vector according to the present invention.

The DNA construct or the recombinant vector of the invention is suitably introduced into a host cell which may be any cell which is capable of expressing the present DNA construct and includes bacteria, yeast and higher eukaryotic cells (Unger, T.F., The Scientist (1 997), 11(1 7), 20-23; Smith, C, The Scientist (1 998), 1 2(22): 20; Smith, C, The Scientist (1 998), 12(3), 1 8; Fernandez, J.M. & Hoeffler, J.P., Gene Expression Systems- using nature for the art of expression, Academic Press 1 999).

Examples of bacterial host cells which, on cultivation, are capable of expressing the DNA construct of the invention are Gram-positive bacteria, eg. species of Bacillus or Gram-negative bacteria such as E. coli. The transformation of the bacteria may be effected by using competent cells in a manner known per se (cf. Sambrook et al., supra).

Examples of suitable mammalian cell lines are the HEK293 and the

HeLa cell lines, primary cells, and the COS (e.g. ATCC CRL 1 650), BHK (eg.

ATCC CRL 1 632, ATCC CCL 10), CHL (e.g. ATCC CCL39) or CHO (eg. ATCC CCL 61 ) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in eg.

Kaufman and Sharp, J. Mol. Biol., (1 982), 1 59, 601 -621 ; Southern and Berg,

J. Mol. Appl. Genet., (1 982), 1, 327-341 ; Loyter et al., Proc. Natl. Acad.

Sci. USA, (1 982), 79, 422-426; Wigler et al., Cell, (1 978), 14, 725; Corsaro and Pearson, Somatic Cell Genetics, ( 1 981 ), 7, 603, Graham and van der Eb,

Virology (1 973), 52, 456; and Neumann et al., EMBO J., (1 982), 1, 841 -

845. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are described, e.g. in US 459931 1 , US 4931 373, US 4870008, US 5037743, and US 4845075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in US 4931 373. The DNA sequence encoding the fluorescent protein of the invention may be preceded by a signal sequence and optionally a leader sequence , e.g. as described above. Further examples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol., (1 986), 1 32, 3459-3465; US 4882279).

Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4745051 ; US 4879236; US 51 55037; US 51 62222; EP 397485, all of which are incorporated herein by reference. The insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5077214). Culture conditions may suitably be as described in, for instance, WO 89/01 029 or WO 89/01 028, or any of the aforementioned references.

In a sixth aspect, the invention provides a method for preparing a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S1 75; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP; the method comprising cultivating a host cell transformed or transfected with a nucleotide sequence according to the invention and obtaining therefrom the polypeptide expressed by said nucleotide sequence.

Suitably, the transformed or transfected host cells as described above are cultured in a suitable nutrient medium under conditions permitting the expression of a DNA construct according to the invention, after which the cells may be used in the screening method of the invention. Alternatively, the cells may be disrupted after which cell extracts and/or supernatants may be analysed for fluorescence and/ or used to purify the GFP or functional GFP analogue of the invention.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published protocols (eg. in catalogues of the American Type Culture Collection; Sambrook et al., supra).

For example, a fusion protein comprising glutathione S-transferase

(GST) and GFP can be constructed and expressed in E. coli. The GFP may be joined in-frame to the C-terminus of GST in a pGEX plasmid vector (Amersham Pharmacia Biotech). Recombinant production of the fusion protein is carried out utilising a standard E. coli expression host, followed by purification employing glutathione affinity chromatography and removal of the GST tag by proteolytic cleavage. ln a seventh aspect of the present invention, there is provided a method of measuring the expression of a protein of interest in a cell. The method comprises: i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue according to the present invention said nucleic acid molecule being operably linked to and under the control of an expression control sequence which moderates expression of said protein of interest; ii) culturing the cell under conditions suitable for the expression of the protein of interest; and iii) detecting the fluorescence emission of the Green Fluorescent Protein (GFP) or a functional GFP analogue as a means of measuring the expression of the protein of interest.

In an eighth aspect of the present invention, there is provided a method of determining the cellular and/or extracellular localisation of a protein of interest which method comprises: i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green

Fluorescent Protein (GFP) or any functional GFP analogue according to the present invention fused to a nucleotide sequence encoding a protein of interest, said nucleic acid molecule being operably linked to and under the control of a suitable expression control sequence; ii) culturing said cell under conditions suitable for the expression of said protein of interest; and iii) determining the cellular and/or extracellular localisation of said protein of interest by detecting the fluorescence emission by optical means.

The fluorescent proteins of the present invention may also be used in a method to detect and compare the effect of a test substance on the regulation of expression and/or translocation of two or more different proteins of interest in a cell. Alternatively, they may be used in a method to compare the expression of a protein of interest and the simultaneous activity of an expression control sequence in response to a test substance. The fluorescent proteins may also be used in a method to compare the activity of two or more expression control sequences in a cell in response to a test substance. Such methods may be performed in the presence and in the absence of a test substance whose effect on the process is to be measured. For example, one detectable reporter molecule may be used as an internal reference and another as a variable marker, since regulated expression of a gene can be monitored quantitatively by fusion of an expression control sequence to a DNA construct encoding, eg. F64L-Y66H-S1 75G-GFP, measuring the fluorescence, and normalising it to the fluorescence of a constitutively expressed spectrally distinct fluorescent molecule. The constitutively expressed spectrally distinct fluorescent molecule, for example GFP, acts as an internal reference.

Thus, in a ninth aspect of the present invention, there is provided a method of comparing the effect of one or more test substance(s) on the expression and/or localisation of one or more different protein(s) of interest in a cell which method comprises: i) introducing into a cell: a) a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue according to the present invention optionally fused to a nucleotide sequence encoding a first protein of interest, said nucleic acid molecule being operably linked to and under the control of a first expression control sequence; and optionally, b) at least one different nucleic acid molecule encoding a protein reporter molecule optionally fused to a different protein of interest, each said nucleic acid molecule being operably linked to and under the control of a second expression control sequence wherei^said protein reporter molecaTeTφjs or is capable of generating an emission signal which is spectrally distinct from that of said fluorescent protein; ii) culturing said cells under conditions suitable for the expression of said protein(s) of interest in the presence and absence of said test substance(s); iii) determining the expression and/or localisation of said protein(s) of interest in said cells by detecting the fluorescence emission by optical means; and iv) comparing the fluorescence emission obtained in the presence and absence of said test substance(s) to determine the effect of said test substance(s) on the expression and/or localisation of said protein(s) of interest.

It will be understood that the method of the ninth aspect may also/optionally be carried out by reference to a database, wherein the response of the cells cultured in the absence of the test substance, in terms of the fluorescence emission measured in step (iii), has already been determined and this value(s) stored upon a database. The fluorescence emission of the cells in the presence of the test substance(s) (as measured in step iv)) is then compared with the value(s) stored on the database in the absence of the test substance(s), to determine the effect of the test substance(s) on the expression and/or localisation of the protein(s) of interest.

In a preferred embodiment of the ninth aspect, samples of said cells in a fluid medium are introduced into separate vessels for each of said test substances to be studied.

Preferably, the first and second expression control sequences are different.

Suitably, the protein reporter molecule may be selected from the group consisting of fluorescent proteins and enzymes. Preferred fluorescent proteins are those which have a spectrally distinguishable emission wavelength compared with the emission wavelength of the fluorescent proteins according to the present invention, for example, BFP. Suitable enzyme reporters are those which are suitable for generating a detectable (eg. a luminescent or fluorescent) signal in a substrate. Suitable enzyme/substrates include: luciferase/luciferin; β-galactosidase/DDAO galactoside; β-galactosidase/fluorescein di-β-D-galactopyranoside; alkaline phosphatase/Attophos.

In the methods of the invention, the fluorescence of cells transformed or transfected with the DNA construct according to the invention may suitably be measured by optical means in for example; a spectrophotometer, a fluorimeter, a fluorescence microscope, a cooled charge-coupled device (CCD) imager (such as a scanning imager or an area imager), a fluorescence activated cell sorter, a confocal microscope or a scanning confocal device, where the spectral properties of the cells in culture may be determined as scans of light excitation and emission.

The fluorescent proteins of the present invention have many additional applications, for example:

i) Use as a non-toxic marker for selection of transfected cells containing an expression vector encoding at least the fluorescent protein of the invention. The fluorescent emission may be used to isolate transfected cells thereby overcoming the need for selection with toxic molecules such as antibiotics.

ii) Use as a protein label in living and fixed cells. The novel proteins exhibit strong fluorescence and are a suitable label for proteins present at low concentrations. Since no substrate is needed and visualization of the fluorescent protein does not damage the cells, dynamic analysis can be performed.

iii) Use as a marker in cell or organelle fusion. By labelling one or more cells or organelles with the novel proteins, for example, F64L-Y66H-S1 75G- GFP, and other cells or organelles with same or another fluor, fusions such as heterokaryon formation can be monitored.

iv) Translocation of proteins fused to the novel proteins of the invention can be visualised. The translocation of intracellular proteins to a specific organelle can be visualised by fusing the protein of interest to a fluorescent protein, for example, F64L-Y66H-S1 75G-GFP, and labelling the organelle with another fluorescent molecule, eg. fluorescent protein. Translocation can then be detected as a spectral shift in the fluorescent proteins in the specific organelle.

v) Use as a secretion marker. By fusion of a fluorescent protein of the invention to a signal peptide or a peptide to be secreted, secretion may be followed in living cells.

vi) Use as genetic reporter or protein tag in transgenic animals. Due to the strong fluorescence of the novel proteins, they are suitable as tags for proteins and gene expression, since the signal to noise ratio is significantly improved over the prior art proteins, such as wild-type GFP.

vii) Use as a cell or organelle integrity marker. By expressing the novel proteins targeted to an organelle, it is possible to calculate the leakage of the protein and use that as a measure of cell integrity.

viii) Use as a transfection marker, and as a marker to be used in combination with FACS sorting (eg. as described in Example 3).

ix) Transposon vector mutagenesis can be performed using the novel proteins as markers in transcriptional and translational fusions. Transposons may be used in microorganisms encoding the novel proteins. The transposons may be constructed for translational and transcriptional fusion to be used for screening for promoters. Transposon vectors encoding the novel proteins, for example, F64L-Y66H-S1 75G-GFP, can be used for tagging plasmids and chromosomes.

x) Use as a reporter for bacterial detection by introducing the novel proteins into the genome of bacteriophages. By engineering the novel proteins, for example, F64L-Y66H-S1 75G-GFP, into the genome of a phage a diagnostic tool can be designed. F64L-Y66H-S1 75G-GFP will be expressed only upon transfection of the genome into a living host. The host specificity is defined by the bacteriophage.

The invention is further illustrated by reference to the following examples and figures in which:

Figure 1 is the nucleotide Sequence of wtGFP (Chalfie et al, Science, (1 994),

263, 802-5) and referred to herein as SEQ ID No.1 . Figure 2 is the corresponding amino acid sequence of wtGFP (Chalfie et al,

Science, ( 1 994), 263, 802-5) and referred to herein as SEQ ID No.2.

Figure 3 is the predicted amino acid sequence of F64L-Y66H-S1 75G-GFP. and referred to herein as SEQ ID No.3.

Figure 4 is a plot showing average fluorescence intensities of mutant BFPs according to the invention.

Figure 5 is a plot showing relative photodegradation of mutant BFPs according to the invention.

EXAMPLES

Cloning of GFP gene and template vector construction

The GFP gene used in the present study was contained within the plasmid pGFP (Chalfie et al., Science, (1 994), 263, 802-805; GenBank accession number U 1 7997) obtained from Clontech Laboratories Inc. (Palo Alto, Ca, USA). The gene was amplified by PCR using Pfu polymerase (Promega, Madison, Wl, USA) according to recognised protocols (Saiki et al., Science, (1 988), 239, 487-491 ). The sequences of primers used were:

GFP-1 5'-ggtacgggccgccaccatgagtaaaggagaagaacttttcac SEQ ID No.4

GFP-2 5'-ggtacgggttaaccggttttgtatagttcatccatg SEQ ID No.5

GFP-3 5'-ggtacgggccgccaccatgggatccaaaggagaagaacttttcac SEQ ID No.6

Primer GFP-1 exhibits homology to the 5' region of the GFP gene and contains a partial Kozak site (Kozak, M, Cell, (1 986), 44, 283) prior to the start codon for efficient initiation of translation in mammalian systems. Primer GFP-2 exhibits homology to the 3' region of the GFP gene and contains an additional Age\ restriction enzyme site immediately prior to the stop codon to facilitate cloning of proteins by fusion to the C-terminus of GFP. Primer GFP-3 is similar to primer GFP-1 exhibiting homology to the 5' region of the GFP gene, but contains an additional restriction site (BamH\) immediately after the initiation codon to facilitate cloning of proteins by fusion to the N-terminus of GFP. Amplified products resulting from PCR reactions containing primers GFP-1 and GFP-2, and GFP-3 and GFP-2 were tailed with a single 3'-deoxyadenosine using Taq polymerase (Amersham Pharmacia Biotech, Amersham, UK) and ligated into the TA cloning vector pTARGET (Promega) according to manufacturer's instructions. The correct orientation relative to the CMV promoter and sequence of the insert was determined by automated DNA sequencing.

2. Generation of mutants of GFP

The following mutants of GFP were generated in the present study: F64L-Y66H-GFP, F64L-Y66H-S1 75G-GFP, F64L-Y66H-V1 63A-GFP.

Mutants of the GFP gene (SEQ ID No.3) construct within pTARGET (See Example 1 ) were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, Ca, USA) according to manufacturer's instructions. The sequences of primers used to generate F64L, V1 63A, S1 75G and Y66H single mutants have been documented in Table 1 . Multiply-mutated GFP molecules were generated through successive mutagenesis reactions. All GFP mutant sequences were verified by automated sequencing.

Table 1

3. Influence of combinations of F64L, Y66H, V1 63A and S1 75G mutations upon BFP when expressed in mammalian cells

Plasmid DNA to be used for transfection was prepared for all BFP constructs using the HiSpeed plasmid purification kit (Qiagen, Westberg, NL). DNA was diluted to 100 ng/μl in 1 8-Megohm water (Sigma) and 1 μg used for transfections. For 50-80% confluency on the day of transfection, HeLa cells were plated at a density of 5x10⁴/well in 6-well plates and incubated overnight. A 1 :3 ( 1 μg : 3 μl) ratio of DNA to FuGeneδ reagent (Roche) was used for each transient transfection reaction; 3 μl FuGeneβ was added to 87 μl serum-free DMEM medium (Sigma) (containing penicillin/streptomycin, L- glutamine (GibcoBRL) and gently tapped to mix, then 1 0 μl (1 μg) construct DNA was added and again gently mixed. The FuGene6:DNA complex was incubated at room temperature for 40 minutes then added dropwise directly to the cells without changing the medium, and the plates swirled for even distribution.

Fluorescence measurements were made 24 hours after transfection.

Briefly, the cells were washed in phosphate-buffered saline, released with the addition of 2 drops of Trypsin (GibcoBRL) and resuspended in 1 ml of complete DMEM medium (containing penicillin/streptomycin, L-glutamine and foetal bovine serum (Sigma). The cells were vortexed and analysed on a FACS Vantage flow cytometer (Becton Dickinson & Co., NJ, USA) for characterisation of whole cell fluorescence, with excitation at 382 nm and emission viewed with fluorescence filter set 424/44 nm (range 402-446 nm). 1 0,000 events were collected for each transfection and fluorescent intensities from the FACS analysis were obtained as geometric means (mean fluorescence on log scale) and are shown in Figure 4.

4. Photodegradation of BFP mutants

To evaluate the relative degree of photodegradation of the BFP mutants, 50ng of DNA was transfected into HeLa cells according to the method outlined in Example 3. For 50-80 % confluency on the day of transfection, HeLa cells were plated at a density of 5x10³/well in a ViewPlate™-96 (Packard, Meriden CT, USA). Twenty-four hours after transfection, the cells were imaged live on a Leadseeker™ Cell Analysis System (Amersham Pharmacia Biotech) and bleached at high laser power

(39.79 mW) with a 364 nm UV laser (emission filter 420-25 nm). Thirty-two individual images were taken over 260 s with non-continuous illumination and all fluorescent proteins showed marked photodegradation as shown in Figure 5.

Claims (1)

1. Claims

   1 . A fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S1 75; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP.

   2. A fluorescent protein according to claim 1 wherein the amino acid F at position 64 has been substituted by an amino acid selected from the group consisting of L, I, V, A and G.

   3. A fluorescent protein according to claim 1 or claim 2 wherein the amino acid Y at position 66 has been substituted by an amino acid selected from H and F.

   4. A fluorescent protein according to any of claims 1 to 3 wherein the amino acid S at position 1 75 has been substituted by an amino acid selected from the group consisting of G, A, L, I and T.

   5. A fluorescent protein according to any of claims 1 to 4 which is F64L- Y66H-S1 75G-GFP.

   6. A fluorescent protein according to any of claims 1 to 5 having an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein having the sequence: SEQ ID No.2.

   7. A fluorescent protein derived from Green Fluorescent Protein (GFP) and having the amino acid sequence as set forth in SEQ ID No.3.

   8. A fusion compound comprising a protein of interest fused to a fluorescent protein said fluorescent protein being a modified protein according to any of claims 1 to 7.

   9. A nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue and has an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein said modified fluorescent protein comprising: i) an amino acid substitution at position F64; ii) an amino acid substitution at position Y66; and iii) an amino acid substitution at position S175; wherein said modified GFP has a different excitation spectrum and/or emission spectrum compared with wild type GFP.

   10. A nucleic acid according to claim 9 encoding a fluorescent protein having an amino acid sequence which is modified by amino acid substitution compared with the amino acid sequence of wild type Green Fluorescent Protein having the sequence: SEQ ID No.2.

   1 1 . The nucleic acid molecule according to claims 9 or 10 encoding a fluorescent protein having an amino acid sequence which is SEQ ID No.3.

   1 2. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein comprising a protein of interest fused to a fluorescent protein according to any one of claims 1 to 7.

   1 3. An expression vector comprising suitable expression control sequences operably linked to a nucleic acid molecule according to any of claims 9 to 1 2.

   1 . A host cell transformed or transfected with a DNA construct comprising an expression vector according to claim 1 3.

   1 5. The host cell according to claim 14 wherein said host cell is selected from the group consisting of a mammalian cell, a bacterial cell, a yeast cell and an insect cell.

   1 6. A method for preparing a Green Fluorescent Protein (GFP) or a functional GFP analogue according to the present invention said method comprising cultivating a host according to claim 14 or claim 1 5 and obtaining therefrom the polypeptide expressed by said nucleotide sequence.

   1 7. A method of measuring the expression of a protein of interest in a cell which method comprises:

   i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue according to any one of claims 1 to 7 said nucleic acid molecule being operably linked to and under the control of an expression control sequence which moderates expression of said protein of interest; ii) culturing said cell under conditions suitable for the expression of said protein of interest; and iii) detecting the fluorescence emission of said Green Fluorescent Protein (GFP) or a functional GFP analogue as a means of measuring the expression of said protein of interest.

   1 8. A method of determining the cellular and/or extracellular localisation of a protein of interest which method comprises:

   i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a fluorescent protein which is derived from Green Fluorescent Protein (GFP) or any functional GFP analogue according to any one of claims 1 to 7 fused to a nucleotide sequence encoding a protein of interest, said nucleic acid molecule being operably linked to and under the control of a suitable expression control sequence; ii) culturing said cell under conditions suitable for the expression of said protein of interest; and iii) determining the cellular and/or extracellular localisation of said protein of interest by detecting the fluorescence emission by optical means.

   1 9. A method of comparing the effect of one or more test substance(s) on the expression and/or localisation of one or more different protein(s) of interest in a cell which method comprises:

   i) introducing into a cell: a) a nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) or a functional GFP analogue according to any one of claims 1 to 7 optionally fused to a nucleotide sequence encoding a first protein of interest, said nucleic acid molecule being operably linked to and under the control of a first expression control sequence; and optionally, b) at least one different nucleic acid molecule encoding a protein reporter molecule optionally fused to a different protein of interest, each said nucleic acid molecule being operably linked to and under the control of a second expression control sequence wherein said protein reporter molecule has or is capable of generating an emission signal which is spectrally distinct from that of said Green Fluorescent Protein (GFP) or functional GFP analogue; ii) culturing said cells under conditions suitable for the expression of said protein(s) of interest in the presence and absence of said test substance(s); iii) determining the expression and/or localisation of said protein(s) of interest in said cells by detecting the fluorescence emission by optical means; and iv) comparing the fluorescence emission obtained in the presence and absence of said test substance(s) to determine the effect of said test substance(s) on the expression and/or localisation of said protein(s) of interest.

   20. The method according to claim 1 9 wherein samples of said cells in a fluid medium are introduced into separate vessels for each of said test substances to be studied.