Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-02-11
2001-07-24
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06265548
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to novel proteins and their production which are useful for detecting gene expression and for visualizing the subcellular targeting and distribution of selected proteins and peptides, among other things. The invention specifically relates to mutations in the gene coding for the jellyfish
Aequorea victoria
green fluorescent protein. (“GFP”), which mutations encode mutant GFP proteins having either an enhanced green or a blue fluorescence, and uses for them.
BACKGROUND OF THE INVENTION
Green fluorescent protein (“GFP”) is a monomeric protein of about 27 kDa which can be isolated from the bioluminescent jellyfish
Aequorea victoria
. When wild type GFP is illuminated by blue or ultraviolet light, it emits a brilliant green fluorescence. Similar to fluorescein isothiocyanate, GFP absorbs ultraviolet and blue light with a maximum absorbance at 395 nm and a minor peak of absorbance at 470 nm, and emits green light with a maximum emission at 509 nm with a minor peak at 540 nm. GFP fluorescence persists even after fixation with formaldehyde, and it is more stable to photo bleaching than fluorescein.
The gene for GFP has been isolated and sequenced. Prasher, D. C. et al. (1992), “Primary structure of the
Aequorea victoria
green fluorescent protein,”
Gene
111:229-233. Expression vectors that comprise the GFP gene or cDNA have been introduced into a variety of host cells. These host cells include: Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293), COS-1 monkey cells, myeloma cells, NIH 3T3 mouse fibroblasts, PtK1 cells, BHK cells, PC12 cells, Xenopus, leech, transgenic zebra fish, transgenic mice, Drosophila and several plants. The GFP molecules expressed by these different cells have a similar fluorescence as the native molecules, demonstrating that the GFP fluorescence does not require any species-specific cofactors or substrates. See, e.g., Baulcombe, D. et al. (1995), “Jellyfish green fluorescent protein as a reporter for virus infections,”
The Plant Journal
7:1045-1053; Chalfie, M. et al. (1994), “Green fluorescent protein as a marker for gene expression,”
Science
263:802-805; Inouye, S. & Tsuji, F. (1994), “Aequorea green fluorescent protein: expression of the gene and fluorescent characteristics of the recombinant protein,”
FEBS Letters
341:277-280; Inouye, S. & Tsuji, F. (1994), “Evidence for redox forms of the Aequorea green fluorescent protein,”
FEBS Letters
351:211-214; Kain, S. et al. (1995), “The green fluorescent protein as a reporter of gene expression and protein localization,”
BioTechniques
(in press); Kitts, P. et al. (1995), “Green Fluorescent Protein (GFP): A novel reporter for monitoring gene expression in living organisms,”
CLONTECHniques
X(1):1-3; Lo, D. et al. (1994), “Neuronal transfection in brain slices using particle-mediated gene transfer,”
Neuron
13:1263-1268; Moss, J. B. & Rosenthal, N. (1994), “Analysis of gene expression patterns in the embryonic mouse myotome with the green fluorescent protein, a new vital marker,”
J. Cell. Biochem
., Supplement 18D Wl6l; Niedz, R. et al. (1995), “Green fluorescent protein: an in vivo reporter of plant gene expression,”
Plant Cell Reports
14:403-406; Wu, G.-I. et al. (1995), “Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins,”
Neuron
14:681-684; Yu, J. & van den Engh, G. (1995), “Flow-sort and growth of single bacterial cells transformed with cosmid and plasmid vectors that include the gene for green-fluorescent protein as a visible marker,” Abstracts of papers presented at the 1995 meeting on “Genome Mapping and Sequencing,” Cold Spring Harbor, p. 293.
The active GFP chromophore is a hexapeptide which contains a cyclized Ser-dehydroTyr-gly trimer at positions 65-67. This chromophore is only fluorescent when embedded within the intact GFP protein. Chromophore formation occurs post-translationally; nascent GFP is not fluorescent. The chromophore is thought to be formed by a cyclization reaction and an oxidation step that requires molecular oxygen.
Proteins can be fused to the amino (N—) or carboxy (C—) terminus of GFP. Such fused proteins have been shown to retain the fluorescent properties of GFP and the functional properties of the fusion partner. Bian, J. et al. (1995), “Nuclear localization of HIV-1 matrix protein P17: The use of
A. victoria
GFP in protein tagging and tracing,”
FASEB J
. 9:AI279; Flach, J. et al. (1994), “A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm,”
Mol. Cell. Biol
. 14:8399-8407; Marshall, J. et al. (1995), “The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function,”
Neuron
14:211-215; Olmsted, J. et al. (1994), “Green Fluorescent Protein (GFP) chimeras as reporters for MAP4 behavior in living cells,”
Mol. Biol. of the Cell
5:167a; Rizzuto, R. et al. (1995), “Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells,”
Current Biol
. 5:635-642; Sengupta, P. et al. (1994), “The
C. elegans
gene odr-7 encodes an olfactory-specific member of the nuclear receptor superfamily,”
Cell
79:971-980; Stearns, T. (1995), “The green revolution,”
Current Biol
. 5:262-264; Treinin, M. & Chalfie, M. (1995), “A mutated acetylcholine receptor subunit causes neuronal degeneration in
C. elegans,” Neuron
14:871-877; Wang, S. & Hazelrigg, T. (1994), “Implications for bcd MRNA localization from spatial distribution of exu protein in Drosophila oogenesis,”
Nature
369:400-403.
A number of GFP mutants have been reported. Delagrave, S. et al. (1995) “Red-shifted excitation mutants of the green fluorescent protein,”
Bio/Technology
13:151-154; Heim, R. et al. (1994) “Wavelength mutations and posttranslational autoxidation of green fluorescent protein,”
Proc. Natl. Acad. Sci. USA
91:12501-12504; Heim, R. et al. (1995), “Improved green fluorescence,”
Nature
373:663-664. Delgrave et al. (1995)
Bio/Technology
13:151-154 isolated mutants of cloned
Aequorea Victoria
GFP that had red-shifted excitation spectra. Heim, R. et al. (1994) “wavelength mutations and posttranslational autoxidation of green fluorescent protein,”
Proc. Natl. Acad. Sci. USA
91:12501-12504 reported a mutant (Tyr66 to His) having a blue fluorescence, which is herein designated BFP(Tyr
67
→His). These references have neither taught nor suggested that their mutations resulted in an increase in the cellular fluorescence of the mutant GFPs.
In general, the level of fluorescence of a protein expressed in a cell depends on several factors, such as number of copies made of the fluorescent protein, stability of the protein, efficiency of formation of the chromophore, and interactions with cellular solvents, solutes and structures. Although the fluorescent signal from wild type GFP or from the reported mutants is generally adequate for bulk detection of abundantly expressed GFP or of GFP-containing chimeras, it is inadequate for detecting transient low or constitutively low levels of expression, or for performing fine structural subcellular localizations. This limitation severely restricts the use of native GFP or of the reported mutants as a biochemical and structural marker for gene expression and morphological studies.
SUMMARY OF THE INVENTION
It an object of the invention to provide engineered GFP-encoding nucleic acid sequences that encode modified GFP molecules having a greater cellular fluorescence than wild type GFP or prior described recombinant GFP.
It is a further object of this invention to provide recombinant vectors containing these modified GFP-encoding nucleic acid sequences, which vectors are capable of being inserted into a variety of cells (including mammalian and eukaryotic cells) and expressing the modified GFP.
It is also an object of this invention to provide host cells capable of providing useful quantities of homogeneous modified GFP.
It is yet another object of this invention to provide peptides that possess a greater cellular fluorescence than native GFP
Gaitanaris George A.
Pavlakis George N.
Stauber Roland H.
Vournakis John N.
Slobodyansky Elizabeth
The United States of America as represented by the Secretary of
Townsend and Townsend / and Crew LLP
LandOfFree
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