Green fluorescent proteins and blue fluorescent proteins

This invention relates to novel fluorescent GFPs and BFPs. A novel BFP according to this invention has an F64L mutation as well as a L236R mutation and is provided with improved fluorescence. Furthermore, another BFP has the F64L mutation with the characteristics as described above and other mutations, V163A and S175G, and it possesses markedly improved characteristics in the expression at 37.degree. C. in addition to those as described above.

BACKGROUND OF THE INVENTION
 1. Field of the invention
 This invention relates to novel fluorescent proteins, GFPs and BFPs.
 2. Related background art
 GFP (Green Fluorescent Protein), which was found in Aequorea victoria, is a
 relatively small protein having a molecular weight of 26,900 and
 comprising the overall 238 amino acid residues as shown below (SEQ No. 1
 in the Sequence Listing).
 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
 1 5 10 15
 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
 20 25 30
 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
 35 40 45
 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe
 50 55 60
 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
 70 75 80
 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
 85 90 95
 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
 100 105 110
 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
 115 120 125
 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
 130 135 140
 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
 145 150 155 160
 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
 165 170 175
 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
 180 185 190
 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
 195 200 205
 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
 210 215 230
 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
 225 230 235 238
 In the present specification, the term "GFP protein" refers to a protein
 that emits green fluorescence when excited by ultraviolet-blue light and
 that, then, does not require an energy source such as a special substrate
 or ATP. In other words, the chromophore formation reaction of GFP is
 autonomous, and the portion of serine-tyrosine-glycine at Nos. 65-67 from
 the amino terminus forms an imidazolidine ring oxidatively which serves as
 a chromophore. (Yuichiro Watanabe, Gendai Kagaku "Modern Chemistry" 12,
 46-52 (1995); R. Heim et al. Proc. Natl. Acad. Sci. USA 91: 12501-12504
 (1994).) Because GFP possesses such a property, a DNA encoding this
 protein is linked to a suitable expression vector and is introduced into
 the desired cells to express GFP, which alone results in fluorescent
 images. Therefore, GFP is in use for the visual analysis of gene
 expression and localization of proteins in a variety of cells in their
 viable state. However, since such GFP was not luminous at 37.degree. C.,
 there was a problem that culturing must necessarily be done at 30.degree.
 C. for the purpose of observation in mammalian cells or the like. In
 connection with this problem, it has been reported that the mutations of
 V163A and S175G enhance the thermal stability. (K. R. Siemering et al.
 Curr. Biol. 6, 1653-1663 (1996).)
 Recently, a mutant of GFP into which the mutations of Y66H and Y145F were
 introduced and which had different wavelength characteristics (it is also
 referred to as "Mutant," and its amino acid sequence is described below
 with the above-mentioned mutations shown as underlined) was developed.
 This is referred to as "BFP (Blue Fluorescent Protein)," because it emits
 blue fluorescence by UV excitation. (R. Heim et al. Curr. Biol. 6, 178-182
 (1996); R. Heim et al. Proc. Natl. Acad. Sci. USA 91, 12501-12504 (1994).)
 In the present specification, the term "BFP protein" refers to a protein
 that emits blue fluorescence when excited by ultraviolet-blue light and
 that, then, does not require an energy source such as a special substrate
 or ATP. However, such BFP had a problem that it experienced severe fading
 as compared to GFP and was difficult to be observed under a microscope or
 the like. As used herein to designate mutation, the position of the
 mutation is expressed by a specific amino acid number in the sequence of
 the above-mentioned wild type; the amino acid prior to its mutation is
 described preceding the number and the mutated amino acid is to be
 described following the number.
 Further, amino acids are designated by the one-letter code or three-letter
 code as appropriate.
 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val (SEQ
 ID NO:2)
 1 5 10 15
 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
 20 25 30
 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
 35 40 45
 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe
 50 55 60
 Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
 66 70 75 80
 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
 85 90 95
 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
 100 105 110
 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
 115 120 125
 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
 130 135 140
 Phe Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
 145 150 155 160
 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
 165 170 175
 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
 180 185 190
 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
 195 200 205
 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
 210 215 230
 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
 225 230 235 238
 SUMMARY OF THE INVENTION
 This invention provides novel fluorescent proteins, GFPs and BFPs.
 The present invention will become more fully understood from the detailed
 description given hereinbelow and the accompanying drawings which are
 given by way of illustration only, and thus are not to be considered as
 limiting the present invention.
 Further scope of applicability of the present invention will become
 apparent from the detailed description given hereinafter. However, it
 should be understood that the detailed description and specific examples,
 while indicating preferred embodiments of the invention, are given by way
 of illustration only, since various changes and modifications within the
 spirit and scope of the invention will become apparent to those skilled in
 the art from this detailed description.
 In view of the above-mentioned problems, the present inventors performed
 extensive research and succeeded in the discovery of novel GFPs and BFPs
 that are free from such problems by introducing certain mutations into
 specific positions of the amino acid sequence for GFP or BFP, thus
 accomplishing this invention.
 Specifically, according to this invention, GFP mutants or BFP mutants were
 prepared from GFP or BFP, either of which was already known (these may be
 hereinafter referred to as "wild type"), by introducing certain mutations
 into its specific positions through various techniques. Then, BFP mutants
 that still emitted brightly after UV radiation for about one hour were
 obtained among such mutants. In other words, the invention has solved the
 problem that the conventional BFP experienced severe fading as compared to
 GFP and was difficult to be observed under a microscope.
 Likewise, a mutant of GFP that was brightly luminous even at 37.degree. C.
 was obtained. Namely, the invention has solved the problem that because
 the conventional GFP was not luminous at 37.degree. C., its observation in
 mammalian cells and the like necessitated the need to culture them at
 30.degree. C.
 Specifically, on the basis of the amino acid sequence for the wild type of
 GFP (283 amino acid residues, SEQ No. 1 in the Sequence Listing), GFPs
 into which the mutations as described below had been introduced were
 prepared, and their fluorescence and thermal characteristics were
 investigated in this invention.
 (1) Phe64Leu
 (2) Val163Ala and Ser175Gly were introduced.
 (3) Phe64Leu, Val163Ala and Ser175Gly were introduced.
 Furthermore, on the basis of the amino acid sequence for the wild type of
 BFP as described above, GFPs into which the mutations as described below
 had been introduced were prepared, and their fluorescence and thermal
 characteristics were investigated in this invention. Here, the mutations
 introduced were based on the amino acid sequence for the wild type of GFP.
 (4) Y66H, Y145F: Phe64Leu, Leu236Arg
 (5) Y66H, Y145F: Phe64Leu
 (6) Y66H, Y145F: Val163Ala, Ser175Gly
 (7) Y66H, Y145F: Phe64Leu, Val163Ala, Ser175Gly, Leu236Arg
 Consequently, it was discovered that the resulting BFP and GFP mutants had
 improved fluorescence characteristics and thermal stability. Specifically,
 this invention provides novel BFPs and GFPs as will be described below,
 and further, genes coding them.
 1. A GFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having at least mutations of
 Phe64Leu, Val163Ala, and Ser175Gly.
 2. A GFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having the three mutations of
 Phe64Leu, Val163Ala, and Ser175Gly.
 3. A BFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having at least mutations of
 Y66H, Y145F, and Phe64Leu.
 4. A BFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having at least mutations of
 Y66H, Y145F, Phe64Leu, and Leu236Arg.
 5. A BFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having the four mutations of
 Y66H, Y145F, Phe64Leu, and Leu236Arg.
 6. A BFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having at least mutations of
 Y66H, Y145F, Phe64Leu, Val163Ala, Ser175Gly and Leu236Arg.
 7. A BFP protein comprising the amino acid sequence set forth in SEQ ID No.
 1 in the Sequence Listing, said sequence having the six mutations of Y66H,
 Y145F, Phe64Leu, Val163Ala, Ser175Gly and Leu236Arg.
 8. A gene encoding the GFP protein according to either Item 1 or Item 2 as
 described above.
 9. A gene encoding the BFP protein according to any of Items 3-7 as
 described above.
 This invention will be illustrated in detail hereinbelow based on its
 embodiments. The abbreviations of nucleic acids and amino acids
 (one-letter and three-letter codes) as used in the present specification
 are set forth below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 Novel GFP or BFP proteins according to this invention are those obtained by
 introducing certain mutations to parts of the amino acid sequences for the
 wild types of GFP and BFP, and exhibit improved fluorescence
 characteristics and thermal stability.
 Therefore, this invention embraces proteins having at. least such amino
 acid sequences insofar as they exhibit the improved fluorescence
 characteristics and thermal stability based on the novel GFP or BFP
 proteins according to the invention. Namely, in the cases where cells of a
 variety of origins are used as will be in use in the Examples below, the
 invention also embraces proteins to which a variety of amino acid
 sequences other than the aforementioned amino acid sequences are appended
 at their N- or C-termini and which exhibit the improved fluorescence
 characteristics and thermal stability based on the novel GFP or BFP
 proteins according to the invention.
 Moreover, this invention provides genes encoding such novel proteins or
 proteins containing them within parts thereof.
 There are no particular limitations to methods for obtaining the novel GFPs
 or BFPs according to this invention, and methods for artificially
 obtaining them by means of chemical syntheses and methods for obtaining
 them according to standard genetic engineering are possible. The latter
 methods are made possible through the genetic engineering techniques in
 which suitable vectors conventionally known and means for introducing
 mutations are combined. Concretely, the following procedure is preferred.
 Specifically, the procedure comprises the steps of: (1) starting with a
 known GFP or BFP protein to be improved and introducing a gene encoding
 said protein into a suitable vector; (2) introducing mutations into said
 gene selectively or randomly according to known methods; and (3) selecting
 desirable mutants on the basis of the fluorescence intensities and
 temperature-dependence, among others, of the resultant GFP or BFP mutants.
 The Contents of Application No.026418/1998, filed on Jan. 23, 1998 in Japan
 is hereby incorporated by reference.
 The above-mentioned procedure will be hereinbelow illustrated in detail by
 way of examples; however, this invention is not to be limited to these
 specific examples.
 EXAMPLES
 (I) The genetic engineering techniques as used in the present examples will
 be illustrated in the following.
 1. Vector Construction
 In this invention, a DNA portion encoding GFP of pGFP-Cl vector (available
 from Clontech Inc.) was replaced by a DNA of GFP derived from phGFP-S65T
 (available from Clontech Inc.), which served as a basic plasmid
 (hereinafter referred to as "phGFP(101)-Cl"). The vector is meant for
 expression in mammalian cells and its full base sequence including the
 vector part is known in the art. The corresponding amino acid sequence is
 set forth below.
 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
 (SEQ ID NO:14)
 1 5 10 15
 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
 20 25 30
 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
 35 40 45
 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe
 50 55 60
 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
 65 70 75 80
 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
 85 90 95
 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
 100 105 110
 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
 115 120 125
 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
 130 135 140
 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
 145 150 155 160
 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
 165 170 175
 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
 180 185 190
 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
 195 200 205
 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
 210 215 230
 Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
 225 230 235 238
 Here, the protein encoded by phGFP-S65T as described above is compared with
 that of a wild type derived from jellyfish: (i) an amino acid (valine) has
 been inserted between methionine, which is amino acid number 1 of the
 amino acid sequence, and serine, which is amino acid number 2; (ii)
 serine, which is amino acid number 65, has been further mutated to
 threonine; and (iii) histidine, which is amino acid number 231, has been
 mutated to leucine. These are respectively underlined in the amino acid
 sequence as described above. Thus for example, the amino acid number 65
 threonine becomes number 66 in reality, but amino acid sequence numbers
 corresponding to those of the wild type are employed for the amino acid
 numbers connected with mutation, in accordance with general rules. In
 other words, the amino acid numbers for the amino acid sequence of the
 wild type derived from jellyfish (amino acid numbers 1 through 238) are to
 be used. The extra valine as described above is construed as having been
 inserted between amino acid number 1 and amino acid number 2, and no
 number is then designated therefor. In practice, such an addition of
 valine has been used as a working example to illustrate the embodiments of
 this invention and it is not the essential amino acid sequence of this
 invention. Accordingly, in the explanation that follows the presence (or
 the absence) of the valine addition will not affect the scope of the
 invention.
 Furthermore, methods for introducing specific mutations are not
 particularly limited, and for example, the method of introduction used in
 the examples of this invention as described below is feasible.
 Specifically, a DNA region encoding GFP was cut out from the
 above-mentioned phGFP(101)-Cl with HindIII, and it was inserted into the
 HindIII site of a pUC18 vector or a pQE30 vector (Qiagen) to thereby
 prepare pUCGFP(101) or pQEGFP(101). Here, the pQE30 vector was meant for
 expression in E. coli.
 Employing the resultant pUCGFP(101), pUCBFP(101) into which the mutations
 of T65S, Y66H, and Y145F had been introduced by the site-directed mutation
 introduction method as described below was prepared.
 Here, through said mutation the amino acid number 65 Ser that was
 introduced by the above-mentioned mutation (T65S) proved to be identical
 with the wild type site.
 Further, a DNA encoding BFP was cut out from the obtained pUCBFP(201) by
 digestion with EcoRI/XhoI and it was cloned into the EcoRI/XhoI site of
 Bluescript II KS(-) (Stratagene) to thereby prepare blueBFP(201).
 Furthermore, a DNA region encoding BFP was cut out from the obtained
 pUCBFP(201) by digestion with HindIII and it was inserted into the HindIII
 site of a pQE30 vector to thereby prepare pQEBFP(201). On the other hand,
 phBFP(201)-Cl was prepared by replacing the GFP coding region of the
 phGFP(101)-Cl vector with the above-mentioned DNA in like manner.
 2. Mutagenic Polymerase Chain Reaction (hereinafter referred to as "PCR")
 Moreover, methods for randomly introducing mutations are not particularly
 limited, and Mutagenic PCR as described below can preferably be used in
 this invention. The Mutagenic PCR can be carried out according to methods
 known in the art. (C. W. Dieffenbach, ed. PCR PRIMER, A Laboratory Manual
 (Cold Spring Harbor Laboratory Press) (1995) pp. 583-588.) Concretely, the
 following conditions were employed in the examples.
 About 50 ng of Plasmid BlueBFP(201) was added to 10.times. mutagenic PCR
 buffer (70 mM MgCl.sub.2, 500 mM KCl, and 100 mM Tris-HCl, pH 8.3 at
 25.degree. C.; 0.1%(w/v) gelatin) 10 .mu.l, 10.times. dNTP (2 mM dGTP, 2
 mM dATP, 10 mM dCTP, and 10 mM dTTP) 10 .mu.l, 10 pmol/.mu.l primer (23mer
 M13Universal primer and M13Reverse primer) 3 .mu.l, and H.sub.2 O 62
 .mu.l, and mixed. Subsequently, 10 .mu.l of 5 mM MnCl.sub.2 was added and
 mixed, and 1 .mu.l of Taq Polymerase (Takara) was added to conduct PCR
 (PC-700 available from ASTEC Inc. was used). The PCR was conducted in
 three tubes under the following conditions: 25 cycles at 94.degree. C. for
 1 min, 30 cycles at 45.degree. C. for 1 min, and 35 cycles at 72.degree.
 C. for 1 min, respectively.
 After the respective reaction solutions were combined and treated with
 chloroform twice, a DNA fragment encoding the amplified BFP was recovered
 by carrying out electrophoresis on a 1% agarose gel after digestion with
 BamHI and XhoI and it was inserted into the BamHI and SalI sites of pQE30
 (Qiagen Inc.).
 Transformation was performed on E. coli JM109, and inoculation was done in
 a LB medium containing carbenicillin to incubate JM109 at 37.degree. C.
 for 16 h. Subsequently, the incubated product was allowed to stand at room
 temperature for 24 h. The E. coli colonies that resulted on a plate were
 irradiated with UV (Funakoshi UV Transilluminator FTI-201 UV 365 nm) from
 the top side of the plate for 1 h, and colonies emitting sufficient
 illumination visually after irradiation were selected: ten colonies were
 obtained in the example.
 Sequence determination was performed on the selected plasmids. With respect
 to the mutant having mutations within its coding region that appeared
 meaningful, the coding region was cut out with Hind3 and was inserted into
 the HindIII site of pQE30, and thereafter, this was cut out with
 SalI/BglII and replaced by the corresponding portion of pQEBFP to bring
 the cloning site of the vector into conformity with pQEBFP(201): in the
 present examples the one prepared from Mutant 10 was designated PQEBFP
 (202).
 3. Construction of Mutant GFP/BFP by the Site-Directed Mutation
 Introduction Method
 The site-directed mutation introduction methods are not particularly
 limited, and for example, the protocol for a Quick Change Kit from
 Stratagene Inc. was followed. The oligonucleotides shown in Table 2 below
 were used as primers and the plasmid (about 0.03 .mu.g) obtained by
 subcloning GFP or BFPcDNA into the HindIII site of a pUC18 or pQE30 vector
 was used as a template. The concrete PCR conditions are preferably as
 follows: 16 cycles at 95.degree. C. for 30 sec, 55.degree. C. for 1 min,
 and 68.degree. C. for 10 min.
 TABLE 2
 oligo
 no. sequence
 1F TCGTGACCACCTTCTCCCACGGCGTGCA (SEQ ID NO:2)
 1R TGCACGCCGTGGGAGAAGGTGGTCACGA (SEQ ID NO:3)
 2F GCTGGAGTACAACTTCAACAGCCACAACG (SEQ ID NO:4)
 2R CGTTGTGGCTGTTGAAGTTGTACTCCAGC (SEQ ID NO:5)
 3F CCTCGTGACCACCCTCTCCCACGGCGTG (SEQ ID NO:6)
 3R CACGCCGTGGGAGAGGGTGGTCACGAGG (SEQ ID NO:7)
 4F CCTCGTGACCACCCTCACCTACGGCGTG (SEQ ID NO:8)
 4R CACGCCGTAGGTGAGGGTGGTCACGAGG (SEQ ID NO:9)
 5F GAACGGCATCAAGGCCAACTTCAAGATCC (SEQ ID NO:10)
 5R GGATCTTGAAGTTGGCCTTGATGCCGTTC (SEQ ID NO:11)
 6F CATCGAGGACGGCGGCGTGCAGCTCGCC (SEQ ID NO:12)
 6R GGCGAGCTGCACGCCGCCGTCCTCGATG (SEQ ID NO:13)
 TABLE 3
 oligo no. used GFP mutant or
 GFP or BFP in the introduction of BFP mutant after
 used as template mutation mutation-introduction
 pUCGFP(101) 1F + 1R pUCGFP101(+Y66H)
 pUCGFP101(+Y66H) 2F + 2R pUC(201)
 pQEGFP(101) 4F + 4R pQEGFP(103)
 pQEBFP(201) 3F + 3R pQEBFP(203)
 pQEGFP(101) 5F + 5R pQEGFP101(+V163A)
 pQEGFP101(+V163A) 6F + 6R pQEGFP(104)
 pQEBFP(201) 5F + 5R pQEBFP201(+V163A)
 pQEBFP201(+V163A) 6F + 6R pQEBFP(204)
 pQEGFP(104) 4F + 4R pQEGFP(105)
 pQEBFP(202) 5F + 5R pQEBFP202(+V163A)
 pQEBFP202(+V163R) 6F + 6R pQEBFP(205)
 Sequence determination of the resulting plasmids was conducted and it was
 verified that the desired mutations were contained in the plasmids.
 In the examples of this invention, GFPs were designated as 101-105 and BFP
 were designated as 201-205 for reasons of convenience to place a variety
 of mutants as obtained in good order. Table 4 below thus summarizes the
 mutations introduced. Although not shown in the table, GFP 101-105 all
 contain the mutations of Ser65Thr and His231Leu.
 TABLE 4
 GFP
 101 none
 103 Phe64Leu
 104 Val163Ala, Ser175Gly
 105 Phe64Leu, Val163Ala, Ser175Gly
 BFP (as for BFP, the two mutations --Tyr66His (Y66H) and Tyr145Phe
 (Y14F)--. have been introduced into the sequenee for GFP
 which serves as a basis)
 201 Y66H, Y145F:
 202 Y66H, Y145F: Phe64Leu, Leu236Arg
 203 Y66H, Y145F: Phe64Leu
 204 Y66H, Y145F: Val163Ala, Ser175Gly
 205 Y66H, Y145F: Phe64Leu, Val163Ala, Ser175Gly, Leu236Arg
 4. Determination of the Quantities of Expression for BFP Mutants
 Determination of the quantities of expression for the BFP mutants obtained
 is not particularly limited, but a comparison of the quantities of their
 expression in E. coli by means of SDS-PAGE is preferable. Concretely, an
 overnight culture of E. coli into which each expression vector of pQE30
 (empty vector), pQEBF(201), and pQEBFP(202) had been introduced was
 diluted to 1/50 and it was grown in 3 ml of 2.times.YT carbenicillin
 medium at 37.degree. C. for 3 h. IPTG was added to each sample to give its
 final concentration of 0.24 mg/ml, and the induction of a BFP protein was
 performed by further culturing the sample for 2.5 h.
 An aliquot (100 .mu.l) was taken out from each sample and centrifuged, and
 precipitates were dissolved in a sample buffer. For each sample, 1.3 ml of
 E. coli was centrifuged at 10,000 rpm for 1 min and precipitates were
 suspended in 26091 of PBS(-). This suspension was frozen and thawed at
 -80.degree. C. for 10 min, and was subjected to ultrasonic treatment (Elma
 Transonic ultrasonic washer 460/H). Subsequently, it was centrifuged at
 15,000 rpm for 5 min to separate soluble proteins from insoluble fractions
 containing the inclusion body. These were subjected to SDS-PAGE in
 quantities that correspond to 50 .mu.l cultures of E. coli and were
 stained with Coomassie Brilliant Blue.
 5. Comparison of Brightness of E. coli Cells Having a Variety of GFPs and
 BFPs Introduced
 JM109 was transformed with each of pQE30 (empty vector), pQEGFP(101),
 pQEGFP(105), pQEBFP(201), pQEBFP(202), and pQEBFP(205), and it was
 streaked on a LB agar medium containing carbenicillin. After incubation at
 37.degree. C. for 24 h, the upper lid was removed and the plate was turned
 upside down and irradiated with UV (Funakoshi UV Transilluminator FTI-201
 UV 365 nm to have photographs taken.
 6. Transfection of GFP and BFP Mutant cDNAs into CHO Cells by the Calcium
 Phosphate Method and Fluorescence Measurements
 A. Transfection
 Coding regions were cut out from the pQE vectors containing the genes of
 GFP and BFP mutants that had been prepared by the site-directed mutation
 introduction method, and the corresponding portions of phGFP(101)-Cl
 vectors were replaced by them; thus, phGFP(103-105)-Cl and
 phBFP(202-205)-Cl were prepared.
 Unless otherwise so stated, CHO-K1 cells were grown in a F12+10% FBS medium
 in 5% CO.sub.2 at 37.degree. C. The cells (1.times.10.sup.5) were
 inoculated into a 6-cm dish, and on the following day, their transfection
 was conducted in two dishes as a pair by the calcium phosphate method. (C.
 Chen and H. Okayama Mol. Cell. Biol. 7: 2745-2752 (1987).) After
 transfection, the one dish was incubated at 37.degree. C. and the other at
 30.degree. C. for 24 h. The transfected CHO cells were washed with
 1.times. PBS(-) three times, and they were dissolved in 1 ml of 10 mM
 Tris-HCl (pH 7.4) containing 1% Triton X-100 and recovered in an Eppendorf
 tube. A supernatant (0.5 ml) from centrifugation at 3,000 rpm for 5 min
 was diluted 4-fold with the same buffer and fluorescence measurement was
 performed. Here, a pUcD2SR.alpha.MCS vector (empty vector) was transfected
 and used as a blank. A Hitachi F-2000 type fluorophotometer was used in
 the fluorescence measurement. In the measurement of GFPs, fluorescence was
 scanned between 460 nm and 600 nm at an excitation wavelength of 460 nm to
 measure the maximal value in the vicinity of the fluorescence wavelength
 of 510 nm. In the measurement of BFPs, fluorescence was scanned between
 360 nm and 500 nm at an excitation wavelength of 360 nm to measure the
 maximal value in the vicinity of the fluorescence wavelength of 445 nm.
 7. Western Blotting
 The CHO cells were transfected with pUcD2SR.alpha.MCS (empty vector)(T.
 Tsukamoto et al. Nature Genet. 11: 395-401 (1995)), phGFP(101)-Cl,
 phGFP(105)-Cl, phBFP(201)-Cl, and phBFP(205)-Cl, respectively and grown at
 37.degree. C. and at 30.degree. C. Employing a sample prior to dilution as
 used in the fluorescence measurement previously described (8 .mu.l),
 SDS-PAGE was performed on a 12% gel. With the use of a Horizonblot (ATTO
 Inc.), transfer was conducted onto a nitrocellulose membrane (Millipore
 Inc., HAHY394FO) under the conditions of 2 mA and 90 min per cm.sup.2.
 After the membrane was taken out and washed with 1.times. PBS, it was
 immersed in 1% skim milk/PBS and shaken at room temperature for 30 min.
 After the membrane was washed with 1.times. PBS, it was immersed in 0.1%
 skim milk/PBS containing an anti-GFP antibody (Clonetech Inc.) that had
 been diluted 2,000-fold and shaken at 4.degree. C. overnight. The membrane
 was washed with 1.times. PBS for 5 min, and then with TPBS (0.05% Trion
 X-100/PBS) for 15 min three times. The membrane was immersed in 0.1% skim
 milk/PBS containing an anti-rabbit IgG antibody labeled with HRP (Amersham
 Inc.) that had been diluted 1,000-fold, and shaken at 4.degree. C. for 1
 h. The membrane was washed with 1.times. PBS for 5 min, and then with TPBS
 (0.05% Trion X-100/PBS) for 15 min three times. The membrane was reacted
 with a chemiluminescence reagent (Amersham Inc. ECL) for 1 min, and then,
 was exposed to an X-ray film for 2 min. (II) Amino Acid Sequences of Novel
 GFP and BFP Mutants
 1. Sequence Determination of BFP Mutants
 Among the 10 mutants obtained, one mutant (Mutant No. 10) proved that
 phenylalanine at amino acid number 64, which had been at the immediate
 N-terminal side of the chromophore, mutated into leucine.
 With respect to this mutant clone, another mutation (L236R) had been
 introduced into its C-terminus (Table 1)
 TABLE 1
 mutant no. mutation
 1 L(CTT)1H(CAT)
 2 D(GAT)7Y(TAT)
 3 I(ATC)6T(ACC)
 4 the multicloning site: 14 bp deletion
 from BamHI
 5 the multicloninq site: 24 bp deletion
 from BamHI
 6 I(ATC)6N(AAC)
 7 L(CTT)4P(CCT), I(ATC)128G(GTC),
 D(GAC)197A(GCC), S(AGC)202C(TGC)
 8 L(CTT)4R(CGT)
 9 M(ATG)1T(ACG), Y(TAC)39N(AAC), K(AAG)52E(GAG)
 10 K(AAG)41K(AAA)silent, F(TTC)64L(CTC),
 L(CTG)236R(CGG)
 With respect to this mutant, BFPcDNA was subcloned into the same HindIII
 site as in pQEBFP(201) for a comparison purpose to prepare pQEBFP(202).
 2. Comparison of the Quantities of Expression for BFP Mutants in E. coli by
 SDS-PAGE
 IPTG was added to E. coli cultures harboring pQEBFP(201) and pQEBFP(202)
 and BFP proteins were allowed to express. When the E. coli cells were
 irradiated with UV, the E. coli harboring pQEBFP(202) apparently exhibited
 stronger fluorescence. When the proteins from these E. coli were analyzed
 by SDS-PAGE, the production of the 31 kDa protein was recognized to almost
 similar degrees in both E. coli having the respective plasmids (FIG. 1,
 Lanes 4 and 7).
 When the solubility of these BFPs was also studied, BFP(201) with weaker
 fluorescence was nearly 3;t insoluble (FIG. 1, Lanes 5 and 6), whereas
 BFP(202) was mostly recovered in the soluble portion (FIG. 1, Lanes 8 and
 9).
 3. Comparison of Fluorescence of E. coil Cells Having a Variety of GFPs and
 BFPs Introduced
 GFPs and BFPs into which the mutations of V163A and S175G had been further
 introduced in addition to F64L were prepared (see Table 4).
 In order to compare the intensities of fluorescence in E. coli, streaking
 was performed using E. coil cells having an empty pQE30 vector or pQE30
 vectors into which cDNAs of GFP101, GFP105, BFP201, BFP202, and BFP205 had
 been subcloned. The E. coli having the empty vector introduced was not
 luminous. The E. coli having BFP201 prior to its improvement subcloned,
 even when irradiated with UV, was hardly luminous. In contrast, the one
 into which 202 had been subcloned was brightly luminous in blue. Further,
 it could be ascertained that 205 was even more brightly luminous than was
 202.
 As for GFPs, green fluorescence was observed by the naked eye, and a
 distinctive difference in brightness was noted between 101 and 105 (FIG.
 2).
 6. Transfection of GFP and BFP Mutant cDNAs into CHO Cells and Fluorescence
 Measurements
 Since very luminous GFPs and BFPs were obtained in E. coli, the comparison
 was made also in mammalian cells (CHO). The results from the fluorescence
 measurements of cell extracts that were already prepared under culturing
 at 37.degree. C. and at 30.degree. C. are summarized (Table 5).
 TABLE 5
 GFP or BFP 37.degree. C. 30.degree. C.
 101 30.8 214.6
 103 532.1 765.4
 104 659.0 697.9
 105 2991.1 868.7
 201 14.3 166.7
 202 304.6 188.6
 203 331.3 210.9
 204 330.9 265.9
 205 901.5 287.7
 The values shown in the table are those obtained by subtracting the value
 of the empty vector used as a blank from the values of fluorescence
 obtained. The blank values were 8.9 in the measurement of GFPs at
 37.degree. C., 7.14 in the measurement at 30.degree. C., 64.3 in the
 measurement of BFPs at 37.degree. C., and 50 in the measurement at
 30.degree. C.
 Table 6 shows relative values when the fluorescence intensity of GFP or BFP
 prior to its improvement after culturing at 37.degree. C. is taken as 100,
 and it also makes comparisons in terms of ratio of fluorescence at
 37.degree. C. to that at 30.degree. C. From Table 6, BFP(202) having the
 mutation as found by the Mutagenic PCR exhibited the fluorescence 21 times
 stronger at 37.degree. C. Further, BFP(202) had two mutations (F64L and
 L236R); however, BFP(203) having only F64L exhibited a similar intensity
 of fluorescence to that of 202. This mutation is believed to have caused
 stronger fluorescence. Seventeen times stronger fluorescence was observed
 in GFP(103) having F64L.
 On the other hand, BFP(204) and GFP(104), both of which had the mutations
 of V163A and S175G, were brighter 23 times and 21 times, respectively.
 GFP(105) and BFP(205) in which these mutations were combined with F64L
 mutation were brighter 97 times and 63 times. In addition, when the ratios
 of fluorescence intensities at 37.degree. C. to those at 30.degree. C. are
 taken for comparison, either of 101 and 201 prior to its improvement was
 darker at 37.degree. C. than at 30.degree. C. Those having F64L alone or
 the combination of V163A and S175G showed increases in the ratio of
 fluorescence intensities at two temperatures, whereas it was found that
 the fluorescence at 37.degree. C. was more than three times brighter with
 respect to GFP(105) and BFP(205) in which the mutations were combined
 (Table 6).
 TABLE 6
 GFP or BFP 37.degree. C. 37.degree. C./30.degree. C.
 101 100 0.14
 103 1728 0.70
 104 2140 0.94
 105 9711 3.44
 201 100 0.09
 202 2130 1.62
 203 2317 1.57
 204 2314 1.24
 205 6304 3.13
 6. Examination of the Quantities of Expression in Animal Cells by Means of
 Western Blotting
 The CHO cells were transfected with pUcD2SR.alpha.MCS (empty vector),
 phGFP(101)-Cl, phGFP(105)-Cl, phBFP(201)-Cl, and phBFP(205)-Cl,
 respectively and cultured at 37.degree. C. and 30.degree. C. Employing an
 anti-GFP antibody for the cultured cells, the quantities of GFP or BFP
 proteins expressed were examined. About 30 kD bands that were not
 recognized in the transfection of the empty vector (FIG. 3, Lanes 1 and 6)
 were detected.
 In culturing at 30.degree. C., no distinctive difference was noted between
 the content of GFP or BFP proteins expressed prior to the introduction of
 mutations and that after the introduction of mutations (Lanes 7-10). On
 the other hand, in culturing at 37.degree. C. it was found that the
 mutants (Lanes 3 and 5) clearly expressed the GFP and BFP proteins in
 larger quantities (FIG. 3, Lanes 2-5).
 The effects associated with the improved BFP and GFP mutants according to
 this invention are summarized below.
 (1) The mutant type BFP(202) obtained by the Mutagenic PCR exhibits
 enhanced fluorescence as compared to BFP prior to the introduction of
 mutation in either E. coli cells or mammalian cells. In the clone of said
 mutant BPF, phenylalanine at amino acid number 64 has mutated into leucine
 (F64L), and further, leucine (amino acid number 236 at the C-terminus) has
 mutated into arginine (L236R).
 With respect to the mutant type BFP(203) having only the mutation at amino
 acid number 64 as described above, a similar enhancement in fluorescence
 was also noted in mammalian cells. Therefore, it is F64L that is the
 responsible mutation for this mutant type BFP(202).
 Such a mutation is presumed to involve a mechanism similar to the
 fluorescence enhancement reported for GFP. (T. -T. Yang et al. Nucleic
 Acids Res. 24: 4592-4593 (1996).)
 (2) The quantities of expression of proteins and the production of soluble
 proteins were investigated: (i) Although the content of proteins is the
 same based on the comparison of the quantities of expression of Mutant
 BFPs in E. coli (through SDS-PAGE), the proteins from the mutant type
 BFP(201) are mostly insoluble whereas soluble proteins have increased in
 the mutant type BPF(202); and (ii) a large difference in brightness was
 also seen in E. coli. These results indicate that the mutant type BPF(201)
 cannot correctly occupy a higher-order structure such as the formation of
 a chromophore whereas the mutant type BPF(202) tends to occupy a more
 correct higher-order structure with ease: the mechanism for the
 above-mentioned fluorescence enhancement is believed to be due to this.
 (3) On the other hand, the results of western blotting in the mammalian
 cells show that the quantity of proteins from GFP or BFP itself has
 increased. Namely, it is thought that the protein can occupy a stabilized
 higher-order structure in the mammalian cells; or alternatively,
 proteolysis becomes slower than that prior to the improvement because the
 protein structure is stabilized.
 (4) With the introduction of the F64L mutation having the characteristics
 as described above and other mutations, V163A and S175G, GFP and BFP
 proteins that have markedly improved characteristics in the expression at
 37.degree. C. in addition to those as described above are obtained.
 Accordingly, the improved types of GFPs and BFPs into which such mutations
 have been introduced are provided with the characteristics that will allow
 them to be brightly luminous even at 37.degree. C., and they will enable
 observation in the mammalian cells where culturing is to be conducted at
 37.degree. C. These improved types of GFPs and BFPs can be applied to cell
 biology as well as to many research areas. FIGS. 4(A) to (B) show the
 effects of this invention as described above.
 From the invention thus described, it will be obvious that the invention
 may be varied in many ways. Such variations are not to be regarded as a
 departure from the spirit and scope of the invention, and all such
 modifications as would be obvious to one skilled in the art are intended
 for inclusion within the scope of the following claims.