Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-02
2003-05-20
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S007200, C435S006120, C435S007800, C435S069100, C435S320100, C435S235100, C530S387100, C530S350000, C530S300000, C536S023100
Reexamination Certificate
active
06566083
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method of detecting biologically active substances affecting intracellular processes, and a DNA construct and a cell for use in the method.
BACKGROUND OF THE INVENTION
Second messengers and protein kinases play key roles in the signalling pathways that control the response of mammalian cells (and probably all eukaryotic cells) to most stimuli. Although such signalling pathways have been subjected to extensive studies, detailed knowledge on e.g. the exact timing and spatial characteristics of signalling events is often difficult to obtain due to lack of a convenient technology. There is, however, one exception to this rule: our understanding of the role of Ca
2+
in e.g. intracellular signalling has been greatly improved due to the development of the fluorescent Ca
2+
probe FURA-2 that permits real times studies of Ca
2+
in single living cells.
Moreover, the construction of probes of cAMP (Adams et al., Nature 349 (1991), 694-697) and activity of the cAMP-dependent protein kinase (Sala-Newby and Campbell, FEBS 307(2) (1992), 241-244) has been attempted. The protein kinase A probe, however, suffers from the drawback that it is based on the firefly luciferase and accordingly produces too little light for fast single cell mesurements. The cAMP probe on the other hand has to be microinjected and is therefore not well suited for routine laboratoy work.In conclusion, both probes lack some of the elegant properties that resulted in the widespread use of FURA-2.
Recently it was discovered that Green Fluorescent Protein (GFP) expressed in many different cell types, including mammalian cells, becam highly flourescent (Chalfie et al., Science 263 (1994), 802-805). WO/07463 describes a cell capable of expressing GFP and a method for selecting cells expressing a protein of interest and GFP based on detection of GFP-flourescence in the cells.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a method of detecting a biologically active substance affecting intracellular processes based on the use of green fluorescent protein, including wild-type GFP derived from the jelly fish
Aequorea victoria
and modifications of GFP, such as modifications that changes the spectral properties of the GFP fluorscence, for the construction of probes, preferably real time probes for second messengers and protein kinase activity.
In one aspect, the present invention relates a DNA construct comprising a DNA sequence coding for
(i) green fluorescent protein (GFP) wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site, or
(ii) a hybrid polypeptide of green fluorscent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site.
In another aspect, the present invention relates to a cell containing a DNA sequence coding for
(i) green fluorscent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition side, or
(ii) a hybrid polypeptide of green fluorescent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site, and capable of expressing said DNA sequence.
In a further aspect, the present invention relates to a method of a detecting a biologically active substance affecting intracellular processes,the method comprising
(a) culturing a cell containing a DNA sequence coding for
(i) green fluorscent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site, or
(ii) a hybrid polypeptide of green flourscent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme reognition site under conditions permitting expression of DNA sequence,
(b) measuring the flourscence of the cell,
(c) incubating the cell with a sample suspected of containing a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorscence measured in step (b), such change being indicative of the prescence of a biologically active substance in said sample.
In a still further aspect, the present invention relates to a method of characterizing the biological activity of a substance with biological activity, the method comprising
(a) culturing a cell containing a DNA sequence coding for
(i) green fluorescent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site or
(ii) a hybrid polypeptide of green fluorescent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site under conditions permitting expression of DNA sequence,
(b) measuring the fluorescence of the cell,
(c) incubating the cell with a sample of a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorscence measured in step (b), said change being characteristic of the biological activity of the biologically active substance in said sample.
Furthermore, studies on the substrate specificity of the different protein kinase A (PKA) isoforms using synthetic peptides have shown that peptides contianing the motifs RRXSX (SEQ ID NO: 33) or RXKRXXSX (SEQ ID NO: 34) (S being the phosphorylated amino acid) tend to be the best substrates for PKA, and a review by Zetterquist, Ö, et al. (in Kemp, B. E. (ed). Peptide and Protein Phosphorylation (1990), 172-188, CRC Press, Boca Raton, Fla., U.S.A.) confirms that most known substrates of PKA contain said motifs.
Available amino acid sequences of GFP do not suggest that GFP is a PKA substrate because of a lack of recognition sites comprising the motifs RRXSX (SEQ ID NO: 33) OR RXKRXXSX (SEQ ID NO: 34). It is therefore surprising that a native or wild-type green fluorescent protein (GFP) derived from the jellyfish
Aequorea victoria
can be phosphorylated by protein kinase A and thereby the spectral properties of GFP are changed resulting in a substantial increase of fluorescence.
In a preferred aspect, the present invention relates to a method of detecting a biologically active substance affecting intracellular processes, the method comprising
(a) culturing a cell containing a DNA sequence coding for a wild-type green fluorescent protein having a protein kinase recognition site under conditions permitting expression of the DNA sequence,
(b) measuring the fluorescence of the cell,
(c) incubating the cell with a sample suspected of containing a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorescence measured in step (b), such change being indicative of the presence of a biologically active substance in said sample.
In a further preferred aspect, the present invention relates to a method of characterizing the biological activity of a substance with biological activity, the method comprising
(a) culturing a cell containing a DNA sequence coding for a wild-type green fluorescent protein having a protein kinase recognition site, under conditions permitting expression of the DNA sequence.
(b) measuring the flourescence of the cell,
(c) incubating the cell with a sample of a biologically active substance affecting intracellular processes, and
(d) measuring the flourescence produced by the incubated cell and determining any change in the flourescence compared to the flourescence measured in step (b), said change being characteristic of the biological activity of the biologically active substance in said sample.
In a still further preferred aspect the present invention relates to
Bjørn Sara Petersen
Poulsen Lars Kongsbak
Thastrup Ole
Tullin Søren
Carlson Karen Cochrane
Novo Nordisk A S
Sredden Sheridan
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