Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1997-10-24
2003-12-09
Kunz, Gary (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S300000, C530S350000, C435S006120, C435S069100, C435S069700, C536S023100
Reexamination Certificate
active
06660844
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the field of cellular signaling, and more specifically to methods for measuring changes that occur during a cell signaling event using optical sensors having fluorescent properties.
FIELD OF THE INVENTION
Fluorescent indicator dyes revolutionized the understanding of cellular physiology by providing a means for continuous measurements in single cells and cell populations. Presently, these dyes are synthesized chemically and introduced as hydrolyzable esters or by microinjection (Cohen, L., and Lesher, S. (1986) Soc. Genl. Physiologists, 40:71-99; Gross, D., and Loew, L. M. (1989) Methods in Cell Biology, 30:193-218; Tsien, R. Y. (1989) Ann. Rev. Neuroscience, 12:227-253). Delivering indicator dyes to specific cell populations has proven to be a difficult problem. In the absence of such cell-specific localization, optical measurement in a tissue cannot distinguish the particular cell population that initiates a signal.
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage, and fusion tags to monitor protein location within cells (e.g., Chalfie, M., et al., Science 263:802-5). Green fluorescent protein (GFP), a small protein, consisting of only 238 amino acids, has been cloned from
Aequorea victoria
(Prasher, D. C., et al. (1992) Gene, 111:229-233). The chromophore of GFP is generated autocatalytically (Heim, R., et al. (1994) Proc. Nat'l. Acad. Sci. USA, 91: 12501-12504), and the protein is stable and functional in many cell types. The crystal structure of GFP has been solved by X-ray diffraction (Ormo, M., et al. (1996) Science, 273:1392-1395; Yang, F., et al. (1996) Nat. Biotechnol., 14:1246-1251). Mutant versions of the green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including different excitation and emission maxima. Fluorescent molecules are attractive as sensors because of the ease of quantification and the highly sensitive nature of the assays. Fluorescent proteins can be produced in vivo by biological systems, and can be used to trace intracellular events. GFP has been shown to fluoresce when expressed from the cDNA, indicating that the protein can undergo the cyclization and oxidation necessary for fluorescence in vivo.
Despite the tremendous advances in the field of genetic engineering and expression of a DNA of interest in a desired cell, few tools exist in the art for the direct, selective measurement of a specific physiological response in a cell. For example, although cell growth can be stimulated during in vitro culture by inclusion of growth factors in the culture medium, there are few adequate means available to directly measure changes in cellular events in response to growth factor administration.
SUMMARY OF THE INVENTION
This invention provides optical sensors for measuring cell signaling or cellular events in vitro or in vivo. The sensors of the invention are chimeric proteins including an optically active polypeptide or fragment thereof, operatively linked to a responsive polypeptide, or responsive fragment thereof. The chimeric protein typically exists in a particular state prior to a cellular event, which state measurably changes during or following the event. Depending on the conformational shape or the state assumed by the expressed protein, a property of the optically active polypeptide changes (e.g., a fluorescence characteristic), thereby making it possible to differentiate between an event before and after it has occurred, and thus an activity of the cell, via the optical output.
In one embodiment, the invention features nucleic acid sequences which encode a chimeric protein including an optically active polypeptide linked to a responsive polypeptide, or responsive fragment thereof, which undergoes a change in response to a cell signaling event and wherein an optical property of the sensor is altered in response to the change. Preferably, the optically active polypeptide or fragment thereof is a fluorescent protein such as GFP.
In another embodiment, the invention features a method for determining the presence of an activity in a sample including contacting the sample with a chimeric protein of the invention; exciting the chimeric protein; and measuring the amount of an optical property in the presence and absence of activity, such that a change in the optical property is indicative of activity. The invention also features a method for determining if a cell exhibits an activity, which includes transfecting a cell with a nucleic acid encoding a chimeric protein of the invention; exciting the chimeric protein; and measuring the amount of an optical property in the presence of the activity and in the absence of the activity, such that a change in the optical property is indicative of activity.
The invention also includes a method for determining whether a compound alters a cell signaling event, including contacting a sample containing a known amount of a cell signaling activity with a test compound and a chimeric protein of the invention; exciting the chimeric protein; measuring the amount of cell signaling activity in the sample as a function of the quantity of change or rate of change of an optical property that differs before and after the cell signaling event; and comparing the amount of activity in the sample with a standard activity for the same amount of the cell signaling event, whereby a difference between the amount of activity in the sample and the standard activity indicates that the compound alters a cell signaling event.
In yet another embodiment, the invention provides a kit for determining the presence of an activity in a sample, including either a chimeric protein of the invention, or a nucleic acid sequence encoding a chimeric protein of the invention. The kit including a nucleic acid encoding a chimeric sensor protein of the invention may provide a host cell stably transfected with the nucleic acid sequence, wherein the cell is now an optical indicator. Thus, the invention also provides a recombinant cell line containing the chimeric protein of the invention. In a specific example provided herein, the responsive polypeptide is a voltage-gated ion channel and the optically active polypeptide is GFP, more specifically, a deletion mutant of GFP. Other modified GFPs are useful in the invention.
Another embodiment of the invention provides a non-human transgenic animal, such as a mouse, which has incorporated into its genome a recombinant nucleic acid sequence encoding a chimeric protein of the invention.
REFERENCES:
patent: 5491084 (1996-02-01), Chalfie et al.
Molloy, M. J et al, The Jellyfish green fluorescent protein:a new tool for studying ion channel expression and function, Neuron, vol. 14, No. 2, pp. 211-215, 1195.*
Kain, S. R. et al., green fluorescent proteins as a receptor of gene expression and protein localization. BioTechniques, vol. 19, No. 4, pp. 650-655, 1995.*
Mannuzzu, L. M. et al., Direct measurement of conformational rearrangement underlying potassium channel gatinf. Science, vol. 271, pp. 213-216, 1996.*
Venerando et al., “Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP)”,FEBS Letter, 382 (1996) 256-260.
Dopf et al., “Deletion mapping of theAequorea victoriagreen fluorescent protein,”Gene, 173 (1996) 39-44.
Htun et al., “Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chmera,”Proc. Natl. Acad. Sci. USA, vol. 3, pp. 4845-4850, May 1996.
Carey et al., “Evidence Using a Green Fluorescent Protein-Glucocorticoid Receptor Chimera that the RAN/TC4 GTPase Mediates an Essential Function Independent of Nuclear Protein Import,”The Journal of Cell Biology, vol. 33, No. 5, Jun. 1996, pp. 985-996.
Girotti et al., “TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possib
Isacoff Ehud Y.
Siegel Micah S.
Basi Nirmal S.
California Institute of Technology
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Kunz Gary
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