Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-04
2004-10-05
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06800437
ABSTRACT:
The present invention is directed to the field of reporter molecules and tags that may be used to monitor a target substance in a biological system. More particularly, the present invention relates to the use of an apo metal binding protein as a reporter molecule or tag. An apo metal binding protein may be bound to a protein or tissue or introduced into a cell. The invention also relates to methods of utilizing the apo metal binding protein for detecting a cell expressing a protein of interest, localizing a protein in a cell, and designing a therapeutic agent for treating a disease or infection.
BACKGROUND
Several methods exist to monitor a target substance in a biological system, including monitoring activities inside a cell and intracellular processes. These methods utilize various reporter molecules and genes and other molecular tags and include, for example, the formation of fusion proteins with coding sequences for chloramphenicol acetyl transferase (CAT), gluceronidase (GUC), beta-galactosidase (bGAL) and luciferases (LUC). Many of these reporters and tags may be used as indicators of processes that would be hard to detect otherwise. Silhavy, T. J. & Beckwith, J. R.
Microbiol. Rev.
(49), 398 (1985); Gould, S. J. & Subramani, S.
Anal. Biochem
. (175), 5 (1998); and Stewart, G. S. A. B. & Williams, P. J.
Gen. Microbiol
. (138), 1289 (1992).
Many uses of these reporter genes have been described extensively in the prior art. There use, however, has been limited because they require extra manipulations. For example, the fixing of cell preparations or the addition of exogenous substrates or cofactors makes it difficult to incorporate the use of these reporters in highly automated assays. In contrast, the use of another reporter gene, the green fluorescent protein (GFP), is not limited by these restrictions but suffers from other disadvantages, such as background noise and the need for complex and expensive equipment for detection. Cormack, B. P., et al.
Gene
, 173(1):33-38 (1996); Kroes, S. J., et al.,
Eur. J. Biochem
. 240(2), 342-351 (1996).
Furthermore, until recently, detection in automated assays was based on enzymatic or fluorimetric methods for reasons of sensitivity. As detection at the microscopic level has developed, however, it has become technically possible to use markers without amplification of the marker itself, or signal amplification. Thus, the development of novel reporter genes or markers that have none of the above limitations and disadvantages are desired.
SUMMARY OF THE INVENTION
To achieve these and other advantages, and in accordance with the purpose of the invention as embodied and broadly described herein, the present invention, in one aspect, provides a method of monitoring a target substance in a biological system comprising providing a biological system with a target substance that is labeled with an apo metal binding protein. Conditions are provided which permit the apo metal binding protein to emit a signal and the signal is observed or measured. The target substance is monitored based on the signal. The method of monitoring a comprise determining the location of the target substance and/or quantifying the amount of target substance in the biological system.
An another embodiment, the invention, using the method of monitoring a target substance described above, provides for a method of determining the cytotoxicity of a drug of interest. The target substance, a cell, is labeled with an apo metal binding protein and is exposed to the drug of interest. The cell is monitored and the cytotoxicity of the drug of interest is determined by whether the cell is influenced by the drug of interest.
The invention also provides for a method of labeling a protein of interest comprising fusing a protein of interest to an apo metal binding protein. In a further embodiment, the invention provides a method of labeling a cell of interest comprising introducing into the cell an apo metal binding protein or by binding to the membrane of the cell an apo metal binding protein.
The invention further provides a method for detecting a cell expressing a protein of interest comprising introducing into the cell a DNA molecule having a DNA sequence encoding the protein of interest and an additional DNA molecule having a DNA sequence encoding an apo metal binding protein. Conditions are then provided which permit expression of the apo metal binding protein and the protein of interest. Conditions are also provided which permit the apo metal binding protein to emit a signal. The cell expressing the protein of interest is detected by observing or measuring the signal of the apo metal binding protein.
In another embodiment, the invention provides a method for localizing a protein of interest in a cell comprising introducing into the cell a DNA molecule comprising a DNA sequence encoding the protein of interest, linked to an additional DNA sequence encoding an apo metal binding protein. The two sequences are linked such that the protein produced by the DNA molecule will have the protein of interest fused to the apo metal binding protein. Conditions are provided which permit expression of the fused protein. Conditions are also provided which permit the apo metal binding protein to emit a signal. The location of the fused protein, and thereby the location of the protein of interest in the cell, is determined by observing or measuring the signal.
The invention also provides a method of designing a therapeutic agent for treating a disease. A cell that is a target of the disease is labeled with the apo metal binding protein and conditions are provided which permit the protein to emit a signal. Whether the therapeutic agent is effective treating the disease is determined by monitoring the signal observed or measured from the cell.
In a further embodiment, the methods of the invention may be used to design a therapeutic agent for treating a virus or bacterial infection comprising labeling the virus or the bacteria with an apo metal binding protein. Conditions are provided which permit the apo metal binding protein to emit a signal and the signal is observed or measured. The effectiveness of the therapeutic agent for treating the virus or bacterial infection is determined by observing or measuring the signal from the virus or the bacteria.
The invention also contemplates a eucaryotic cell comprising a DNA sequence that encodes an apo metal binding protein. In one embodiment, the eucaryotic cell is an animal cell, the apo metal binding protein is a copper binding protein and the DNA sequence comprises the azu gene.
Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements are combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
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Cormack et al., “FACS-optimized Mutants of the Green Fluorescent Protein (GFP),”Gene, vol. 173 pp., 33-38 (1996).
Garrett et al., “The Crystal Structure of Poplar Apoplastocyanin at 1.8-Å Resolution,”The Journal of Biological Chemistry, vol. 259, No. 4, pp. 2822-2825 (1984).
Garrett et al., “Type-1 Copper (Blue) Proteins Signature,” http://bess.u-strasbg.fr/BioInfo/Proste/00174.pdoc (Last Update: 1995).
Holm et al., “Structural and Functional Aspects of Metal Sites in Biology,”Chem. Rev., vol. 96, pp. 2239-2314 (1996).
Kroes et al., The Mutation Met121→His Crea
De Kerpel Jan Octaaf Antoon
Dierynck Inge
Pauwels Rudi Wilfried Jan
Roelant Christiaan Hubert Simon
Van Acker Koenraad Lodewijk August
McKelvey Terry
Tibotec Bvba
Woodcock & Washburn LLP
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