Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-04-01
2002-01-29
Brusca, John S. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S320100, C435S325000, C530S350000, C536S023200, C536S023400, C536S023500, C536S023700
Reexamination Certificate
active
06342345
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of molecular biology and, more specifically, in the field of reporter systems useful for the analysis of protein-protein interactions.
BACKGROUND
The &bgr;-galactosidase enzyme (&bgr;-gal), the protein product of the
E. coli
lacZ gene, is widely used in studies of gene expression and cell lineage in higher organisms. Several biochemical assays of &bgr;-gal activity, including live-cell flow cytometry and histochemical staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl &bgr;-D-galactopyranoside (X-gal) make the product of the lacZ gene extremely versatile as a quantitative reporter enzyme, selectable marker, or histological indicator. Bronstein et al. (1989)
J. Biolumin. Chemilumin.
4:99-111; Nolan et al. (1988)
Proc. Natl. Acad. Sci. USA
85:2603-2607; and Lojda (1979)
Enzyme Histochemistry: A Laboratory Manual
, Springer, Berlin. One property of the lacZ system that has been well-characterized in studies of bacterial genetics, but has not been exploited in eukaryotes is the phenomenon of intracistronic complementation. Studies in
E. coli
have shown that deletions of &bgr;-gal which remove portions of either the N-terminus or the C-terminus produce enzyme which is inactive. However, coexpression of one of these deletion mutants with a second inactive deletion mutant containing domains that are lacking in the first can restore &bgr;-gal enzymatic activity in a process called complementation. This complemented &bgr;-gal activity arises by concentration-dependent assembly of a stable hetero-octameric enzyme complex comprising all the essential domains of the wild-type homotetramer. Ullman et al. (1965)
J. Mol. Biol
12:918-923; Ullman et al. (1967)
J. Mol. Biol
24:339-343; and Ullman et al. (1968)
J. Mol. Biol.
32:1-13.
A system utilizing &bgr;-gal complementation in enzyme assays has been described. Henderson, U.S. Pat. No. 4,708,929. In this system, enzymatically inactive &bgr;-gal polypeptide fragments, capable of combining with high affinity to form active &bgr;-gal by complementation, are used. One of the fragments is conjugated to analyte, which allows it to compete with analyte for binding to an analyte-binding protein. If bound to the analyte-binding protein, the &bgr;-gal fragment is unable to complement. Thus, by comparing &bgr;-gal activity in the presence of sample to that obtained in the presence of a known concentration of analyte (at equal concentrations of analyte-binding protein) the amount of analyte in the sample may be determined. This method requires high-affinity complementing subunits of &bgr;-gal, requires that an analyte-binding protein be known, and is not applicable to single-cell analysis.
Previous systems for the study of protein-protein interactions have been described which utilize two fusion genes whose products reconstitute the function of a transcriptional activator. Fields et al., (1989)
Nature
340:245-247; Bai et al., (1996)
Meth. Enzymol.
273:331-347; Luo et al., (1997)
BioTechniques
22(2):350-352. In one fusion gene, a sequence encoding a first protein is conjugated to a sequence encoding a DNA-binding domain of a transcriptional regulatory protein. In a second fusion gene, a sequence encoding a second protein is conjugated to a sequence encoding a transcriptional activation domain of a transcriptional regulatory protein. The two fusion genes are co-transfected into a cell which also contains a reporter gene whose expression is controlled by a DNA regulatory sequence that is bound by the DNA-binding domain encoded by the first fusion gene. Expression of the reporter gene requires that a transcriptional activation domain be brought adjacent to the DNA regulatory sequence. Binding of the first protein to the second protein will bring the transcriptional activation domain encoded by the second fusion gene into proximity with the DNA-binding domain encoded by the first fusion gene, thereby stimulating transcription of the reporter gene. Thus, the level of expression of the reporter gene will reflect the degree of binding between the first and second proteins.
There are several disadvantages associated with the use of the above-mentioned system. As it is dependent upon transcriptionally-regulated expression of a reporter gene, this system is limited to the assay of interactions that take place in the nucleus. In addition, the assay is indirect, relying on transcriptional activation of a reporter gene whose product is diffusible. Hence, a method which would allow a direct and immediate examination of molecular interactions, at the site where they occur, would be desirable.
A system for detecting protein-protein interactions, not limited to nuclear interactions, has been described. U.S. Pat. Nos. 5,503,977 and 5,585,245. In this system, fusions between potential interacting polypeptides and mutant subunits of the protein ubiquitin are formed. Juxtaposition of the two ubiquitin subunits brought about by interaction between potential interacting polypeptides creates a substrate for a ubiquitin-specific protease, and a small peptide reporter fragment is released. In this system, binding between the potential interacting polypeptides does not generate any type of enzymatic activity; therefore, signal amplification is not possible. Additionally, the ubiquitin system does not measure activity in intact cells, but relies on assays of proteolysis in cell-free extracts. What is needed is a sensitive method for examining protein interactions in intact cells in the relevant cellular compartment.
Fluorescence imaging has been used to study the intracellular biochemistry of living cells. A fluorescent indicator for the adenosine 3′,5′-cyclic monophosphate (cAMP) signaling pathway has been described in which the sensor is a cAMP kinase in which the catalytic and regulatory subunits each are labeled with a different fluorescent dye, such as fluorescein or rhodamine, capable of fluorescence resonance energy transfer in the holoenzyme complex. A change in shape of the fluorescence emission spectrum occurs upon cAMP binding, and therefore activation of the kinase can be visualized in cells microinjected with the labeled holoenzyme. Adams et al.,
Nature,
349: 694-697 (1991). This system is limited by the fact that it requires microinjection, and a preferred distance between the labeled units for energy transfer to occur.
Substrates for &bgr;-lactamase have been described in the art which include a fluorescent donor moiety and a quencher, which include an attached group which makes them permeable through cell membranes, wherein the attached group is hydrolyzed off after the substrate enters the cell. Fluorescence energy transfer between the donor and quencher is monitored as an indicator of &bgr;-lactamase activity. This system also can be used in a reporter gene assay using cells containing &bgr;-lactamase reporter genes functionally linked to a promoter. PCT WO 96/30540 published Oct. 3, 1996, the disclosure of which is incorporated herein.
DISCLOSURE OF THE INVENTION
The present invention provides methods and compositions for detecting, assaying and quantitating molecular interactions within living cells and in vitro, through complementation between two or more low affinity reporter subunits, such as distinct
E. coli
lacZ mutations. In a preferred embodiment, protein-protein interactions within living cells are detected and quantitated using the methods and compositions of the present invention. The practice of the present invention enables, for the first time, the study of protein-protein interactions and their control in living mammalian cells without reliance upon the transcriptional activation of a reporter gene construct. Association of the proteins of interest results directly in enzyme activity and is independent of other cellular functions. Therefore, the present invention provides advantages over other systems currently in use by allowing the detection of complexes that are excluded from the nucleus, and detection of complexes whose formation would inhibit tra
Blau Helen M.
Mohler William
Rossi Fabio
Brusca John S.
Morrison & Foerster / LLP
The Board of Trustees of the Leland Stanford Junior University
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