Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-10-15
2002-07-02
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S189000
Reexamination Certificate
active
06414119
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of biotechnology research products, fluorescent proteins and microscopy.
BACKGROUND OF THE INVENTION
Various scientific articles are referred to in parentheses throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The green fluorescent proteins (GFPs) are a unique class of chromoproteins found in many bioluminescent hydrozoan and anthozoan coelenterates, including the hydromedusan jellyfish (
Aequoria victoria
). The gene for
A. victoria
GFP has been cloned into procaryotic and eucaryotic hosts and is expressed in such organisms as a functional fluorescent protein with spectral characteristics identical to the native
A. victoria
GFP.
Mutants of
A. victoria
GFP with altered spectral properties have been isolated or created. For instance, a Tyr
66
His mutant (Heim et al., Proc. Natl. Acad. Sci. USA 91: 12501-12504, 1994) displays blue fluorescence instead of green, and a Ser
65
Thr mutant (Heim et al., Nature 373: 663-664, 1994) displays a red-shifted absorption peak. A set of mutants within and surrounding the chromophore has been disclosed (Cormack et al., Gene 173: 33-38, 1996), which have shifted excitation spectra and possible improved folding properties, as compared with wild-type GFP.
In nature, GFP from
A. victoria
converts would-be blue bioluminescence from excited aequorin to green (&lgr;max=509), identical in color to its own fluorescence. The GFP chromophore is formed by post-translational cyclization of an internal peptide, Ser65-deHTyr-Gly. Though the chemical mechanism for chromophore formation is not fully understood, it is clear that molecular oxygen is required. It also appears that chromophore formation is an autocatalytic event, requiring no cofactors or enzymes other than the GFP itself.
Chromophore formation in GFP is relatively slow, occurring with a time constant of approximately 4 hours. Variants have been created or selected in
E. coli
with post-translational chromophore formation rates of less than two hours. For instance, the “cycle 3” variant (Crameri et al., Nature Biotech. 14: 315, 319, 1996), reaches 50% of maximum fluorescence in
E. coli
in 95 minutes following induction of expression. However, chromophore formation even in this mutant is dependent on the presence of molecular oxygen. In low oxygen, chromophore formation is retarded, as is the case with wild-type GFP.
The requirement for molecular oxygen for chromophore formation in GFP limits the utility of cloned GFP and GFP mutants for bioluminescent labeling under low-oxygen conditions, such as often exist in cells or tissues in situ and in vivo. The use of GFP is also limited to the range of environmental conditions in which wild type GFP is stable. Accordingly, a need exists to create or select for GFP mutants capable of chromophore formation in low oxygen and with increased stability.
SUMMARY OF THE INVENTION
In accordance with the invention, a new category of green fluorescent protein variant is provided. Several single amino acid changes around the chromophore of the protein are described that give the GFP the novel characteristic of rapid chromophore formation under low oxygen conditions as examples of this new category. This category of variant also has the benefits of rapid chromophore formation under conditions of normal oxygen and surprising stability.
One aspect of the invention is a new category of GFP variant in which the chromophore forms at low it oxygen concentrations. In a preferred embodiment, this GFP variant also has more rapid chromophore formation than the wild type GFP at normal oxygen conditions. In a most preferred embodiment, the variant is also more thermotolerant than the wildtype GFP.
Another aspect of the invention is a variant in GFP protein that comprises a native GFP protein with at least one mutation, where the mutation is selected from F64C, F64M and V68C; and the GFP protein is at least 80% similar to SEQ ID NO:1. In a preferred embodiment, the variant protein additionally has a mutation selected from Y66H and S65T. This embodiment also include antibodies immunologically specific to peptides comprising the variant amino acids at positions 64 or 68.
Another aspect of the invention encompasses nucleic acid sequences that encode the proteins of the invention. In a preferred embodiment, the nucleic acid encodes a cysteine in positions 64 or 68, or a methionine in position 64 and is at least 60% identical to SEQ ID NO:2, or hybridizes at moderate stringency to SEQ ID NO:2. In a more preferred embodiment, the sequence is SEQ ID NO:2 except that a cysteine is encoded in positions 64 or 68, or a methionine is encoded in position 64. In a most preferred embodiment, the sequence additionally encodes for histidine at position 66 or threonine at position 65. This aspect of the invention includes oligonucleotides that comprise the nucleic acids that encode the variant amino acids at positions 64 or 68.
Another aspect of the invention is a fusion protein comprising the proteins of the invention fused to a His-tag. In a preferred embodiment, the His-tag is fused to the carboxy-terminus of the protein. Any more preferred embodiment, the His-tag is fused to the carboxy side of the histidine 231 via a linker peptide. In a most preferred embodiment, the peptide of SEQ ID NO:3 is fused to the carboxy side of histidine 231. This aspect further includes isolated nucleic acid molecules that encode the fusion protein, and the nucleic acids operably inserted into a vector for replication in cells.
Another aspect of the invention is an expression cassette comprising a coding sequence for the protein of the invention. In a preferred embodiment, the expression cassette comprises the nucleic acid of the invention. In another preferred embodiment, the expression cassette encodes the His-Tag-variant GFP fusion protein of the invention. In more preferred embodiment, the expression cassette comprises regulatory sequences suitable for expression in plant, animal or bacterial cells. In a more preferred embodiment, the expression cassette is operably inserted into a vector for stable transformation of bacterial, plant or animal cells. This aspect of the invention also includes cells transformed with the expression cassette, and plants and animals regenerated from the transformed cells.
REFERENCES:
R. Heim, et al. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein. (1994)Proc. Natl. Acad. Sci.91:12501-12504.
R. Heim, et al. Improved Green Fluorescence. (1995)Nature373:663-664.
B.P. Cormack, et al. FACS-Optimized Mutants of the Green Fluorescent Protein (GFP). (1996)Gene173:33-38.
A. Crameri, et al. Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling. (1996)Nature Biotechnology. 14:315-319.
S. Delagrave, et al. Red-Shifted Excitation Mutants of the Green Fluorescent Protein. (1995)Bio/technology. 12:151-154.
B.G. Reid and G.C. Flynn. Chromophore Formation in Green Fluorescent Protein. (1997)Biochemistry. 36:6786-6791.
F. Yang, et al. The Molecular Structure of Green Fluorescent Protein. (1996)Nature Biotechnology. 14:1246-1251.
M. Ormo, et al. Crystal Structure of the Aequorea Victoria Green Fluorescent Protein. (1996)Science. 273:1392-1395.
W. Ward.Biochemical and Physical Properties of Green Fluorescence Protein. 1998. 45-75.In Green Fluorescent Protein: Properties, Applications and Protocols, M. Chalfie and S. Kain, eds., Willey-Liss.
Palm et al. (May 1997) Nature Structural Biology, vol. 4(5), pp. 361-365.
Licata & Tyrrell P.C.
Rutgers, the State University
Slobodyansky Elizabeth
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