Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-30
2001-10-23
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S070100, C435S320100, C435S325000, C435S069100, C435S455000, C530S350000, C530S324000, C536S023400, C536S023500
Reexamination Certificate
active
06306600
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of biochemical assays and reagents. More specifically, this invention relates to modified fluorescent proteins and to methods for their use.
2. Description of the Related Art
Because of its easily detectable green fluorescence, green fluorescent protein (GFP) from the jellyfish
Aequorea Victoria
has been used widely to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in numerous species and in a wide variety of cells. GFP is a very stable protein which can accumulate and thus is often toxic to mammalian cells.
Recently, crystallographic structures of wild-type GFP and the mutant GFP S65T reveal that the GFP tertiary structure resembles a barrel (Ormo et al. (1996)
Science
273: 1392-1395; Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996)
Nature Biotech
14: 1246-1251). The barrel consists of beta sheets in a compact antiparallel structure. In the center of the barrel; an alpha helix containing the chromophore is shielded by the barrel. The compact structure makes GFP very stable under diverse and/or harsh conditions, such as protease treatment, making GFP an extremely useful reporter in general. On the other hand, its stability makes it difficult to determine short-term or repetitive events.
A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful for a variety of research purposes. New versions of GFP have been developed via mutation, including a “humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (see Cormack, et al., (1996)
Gene
173, 33-38; Haas, et al., (1996)
Current Biology
6, 315-324; and Yang, et al., (1996)
Nucleic Acids Research
24, 4592-4593). One such humanized protein is “enhanced green fluorescent protein” (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-fluorescent light emitting versions.
Ornithine decarboxylase (ODC) is an enzyme critical in the biosynthesis of polyamines. Murine ornithine decarboxylase is one of most short-lived proteins in mammalian cells, with a half life of about 30 minutes (Ghoda, et al., (1989)
Science
243, 1493-1495; and Ghoda, et al. (1992)
Mol. Cell. Biol
. 12, 2178-2185). Rapid degradation of murine ornithine decarboxylase has been determined to be due to the unique composition of its C-terminus, a portion of which has a PEST sequence—a sequence which has been proposed as characterizing short-lived proteins. The PEST sequence contains a region enriched with proline (P), glutamic acid (E), serine (S), and threonine (T), often flanked by basic amino acids, lysine, arginine, or histidine (Rogers, et al., (1989)
Science
234:364-68; Reichsteiner, M. (1990) Seminars in
Cell Biology
1:433-40).
The ornithine decarboxylase of Trypanosoma brucei (TbODC) does not have a PEST sequence, and is long-lived and quite stable when it is expressed in mammalian cells (Ghoda, et al. (1990)
J. Biol. Chem
. 265: 11823-11826); however appending the C terminus of murine ornithine decarboxylase to TbODC makes TbODC become unstable. Moreover, deletion of the C-terminal, PEST-containing region from murine ornithine decarboxylase prevents its rapid degradation (Ghoda, L., et al. (1989)
Science
243: 1493-1495).
The prior art is deficient in a destabilized or short-lived GFP. The present invention fulfills this need in the art.
SUMMARY OF THE INVENTION
A rapid turnover or destabilized GFP can be used in research applications where prior art GFPs cannot. Such applications include using the destabilized GFP as a genetic reporter for analyzing transcriptional regulation and/or cis-acting regulatory elements, or as a tool for studying protein degradation. Further, a rapid turnover GFP permits easier development of stable cell lines which express the GFP gene, since toxic levels of GFP are avoided because the GFP protein is degraded quickly.
The present invention provides a fusion protein with a half life decreased markedly from that of wildtype GFP. In one embodiment, there is provided a fusion protein comprising an EGFP fused to a peptide producing a destabilized protein. In another embodiment, there is provided a fusion protein with a half life of about ten hours or less, preferably with a half life of about 4 hours or less, more preferably with a half life of 2 hours or less and even more preferably with a half life of 1 hour or less. A preferred embodiment of this aspect of the invention includes EGFP, and/or a PEST sequence-containing portion of a C-terminus of murine ornithine decarboxylase (MODC). Specific preferred embodiments of the present invention include EGFP-MODC
376-461
; EGFP-MODC
376-456
; EGFP-MODC
422-461
; P426A/P427A; P438A; E428A/E430A/E431A; E444A; S440A; S445A; T436A; D433A/D434A; and D448A.
In yet another aspect of the invention, there is provided an isolated DNA molecule encoding a fluorescent fusion protein with a half life that is markedly decreased from that of wildtype GFP. In one embodiment of this aspect of the invention, there is provided a n isolated DNA molecule encoding a fluorescent fusion protein with a half life of about ten hours or less, preferably with a half life of about 4 hours or less, more preferably with a half life of 2 hours or less and even more preferably with a half life of 1 hour or less. In a preferred embodiment of this aspect of the invention, the isolated DNA molecule encoding the fluorescent fusion protein is a synthetic GFP gene containing codons preferentially found in highly expressed human proteins. Further, the present invention provides a vector capable of expressing the isolated DNA molecule encoding a GFP fusion protein with a decreased half life. In one embodiment of the vector, the vector contains an inducible promoter.
In another aspect of the invention, there is provided a method of labeling cells with a transient GFP reporter. In this method, a DNA vector comprising an inducible promoter and the isolated DNA encoding a GFP fusion protein with a decreased half life is utilized. This vector is transfected into cells which are cultured under conditions in which the promoter induces transient expression of the GFP fusion protein of the present invention, which provides a transient fluorescent label.
REFERENCES:
Rechsteiner et al., Cell Biology, vol. 1, pp. 435-440, 1990.*
Loetscher et al., The Journal of Biological Chemistry, vol. 266, No. 17, pp. 11213-11220, 1991.*
Li et al., The Journal of Biological Chemistry, vol. 273, No. 52, pp. 34970-34975, Dec. 25, 1998.*
Moradpour et al., Characterization of Cell Lines Allowing Tightly Regulated Expression of Hepatitis C Virus Core Protein, Virology vol 222, p. 51-63, 1996.
Kain Steve
Li Xianqiang
Bozicevic Field & Francis LLP
Caputa Anthony C.
Clontech Laboratories, Inc.
Field Bret E.
Hunt Jennifer
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