Green fluorescent proteins and blue fluorescent proteins

Details Green fluorescent proteins and blue fluorescent proteins Green fluorescent proteins and blue fluorescent proteins

1998-07-24

2001-02-27

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S440000, C536S023100

Reexamination Certificate

active

06194548

ABSTRACT:

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° C., there was a problem that culturing must necessarily be done at 30° 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 describ

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