Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-03-14
2001-03-06
Scheiner, Laurie (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069700, C435S069100
Reexamination Certificate
active
06197928
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to fluorescent protein sensors for detecting and quantifying analytes.
Measurement of an analyte concentration in vitro or in vivo by non-invasive techniques can help elucidate the physiological function of the analyte. This can also aid in identifying changes that occur in a cell or organism in response to physiological stimuli. For example, cyclic AMP can be detected by fluorescence resonance energy transfer between a separately labeled proteins that associate with each other but are not covalently attached to each other. See, U.S. Pat. No. 5,439,797.
For example, many effects of Ca
2+
in cells are mediated by Ca
2+
binding to calmodulin (CaM), which causes CaM to bind and activate target proteins or peptide sequences. Based on the NMR solution structure of CaM bound to the 26-residue M13 Ca
2+
-binding peptide of myosin light-chain kinase, Porumb et al. fused the C-terminus of CaM via a Gly—Gly spacer to the M13. Ca
2+
binding switches the resulting hybrid protein (CaM-M13) from a dumbbell-like extended form to a compact globular form similar to the CaM-M13 intermolecular complex. See, Porumb, T., et al.,
Prot.Engineering
7:109-115 (1994).
Fluorescent Ca
2+
indicators such as fura-2, indo-1, fluo-3, and Calcium-Green have been the mainstay of intracellular Ca
2+
measurement and imaging. See, for example, U.S. Pat. Nos. 4,603,209 and 5,049,673. These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents. Although the Ca
2+
-indicating photoprotein aequorin is targetable, the photoresponse to Ca
2+
is low since it is chemi-luminescent. Moreover, aequorins need to incorporate exogenous coelenterazine.
SUMMARY OF THE INVENTION
This invention provides fluorescent indicators and methods for using them to determine the concentration of an analyte both in vitro and in vivo. In one aspect, the fluorescent indicator includes a binding protein moiety, a donor fluorescent protein moiety, and an acceptor fluorescent protein moiety. The binding protein moiety has an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte. The donor fluorescent protein moiety is covalently coupled to the binding protein moiety. The acceptor fluorescent protein moiety is covalently coupled to the binding protein moiety. In the fluorescent indicator, the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited.
The donor fluorescent protein moiety and the acceptor fluorescent protein moiety can be Aequorea-related fluorescent protein moieties. Preferably, the donor fluorescent protein moiety is P4-3, EBFP, or W1B, and the acceptor fluorescent protein moiety is S65T, EGFP, or 10c.
In preferred embodiments, the indicator further includes the target peptide moiety and a linker moiety that covalently couples the binding protein and the target peptide moiety. The binding protein moiety further includes a peptide-binding region for binding the target peptide moiety. The binding protein moiety can be covalently coupled to the donor fluorescent protein moiety and the target peptide moiety can be covalently coupled to the acceptor fluorescent protein moiety.
The indicator can be a single polypeptide. In preferred embodiments, one of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is covalently coupled to the carboxy terminus of the single polypeptide and the other of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is covalently coupled to the amino terminus of the single polypeptide.
The indicator can include a localization sequence. The localization sequence can be a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial import sequence, a mitochondrial localization sequence, or a localized protein.
In preferred embodiments, the linker moiety is a peptide moiety. The linker moiety can include between about 1 amino acid residue and about 20 amino acid residues. The linker moiety can be -Gly—Gly-.
Preferably, the binding protein moiety is calmodulin, a calmodulin-related protein moiety, cGMP-dependent protein kinase, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, protein kinase C, inositol-1,4,5-triphosphate receptor, or recoverin. A calmodulin-related protein moiety is derived from calmodulin that has been modified to have a different binding affinity for calcium or a target peptide moiety.
Most preferably, the binding protein moiety is calmodulin or a calmodulin-related protein moiety. In these embodiments, the target peptide moiety can be a subsequence of a calmodulin-binding domain of M13, smMLCKp, CaMKII, Caldesmon, Calspermin, Calcineurin, PhK5, PhK13, C28W, 59-kDa PDE, 60-kDa PDE, NO-30, AC-28,
Bordetella pertussis
AC, Neuro-modulin, Spectrin, MARCKS, F52, &bgr;-Adducin, HSP9Oa, HIV-1 gp160, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, or Model Peptide CBP2. Preferably, the target peptide moiety is M13.
In another aspect, the invention features a fluorescent indicator including a target peptide moiety, a binding protein moiety, a linker moiety, a donor fluorescent protein moiety covalently coupled to the binding protein moiety, and an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety. The binding protein moiety has an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte. The linker moiety covalently couples the binding protein and the target peptide moiety and is a peptide moiety. The binding protein moiety has a peptide-binding region for binding the target peptide moiety. The indicator is a single polypeptide.
In another aspect, the invention features a method for determining the concentration of an analyte in a sample. The method includes the steps of contacting the sample with a fluorescent indicator having a donor fluorescent protein moiety, binding protein moiety, and acceptor protein moiety, exciting the donor moiety, and determining the degree of fluorescence resonance energy transfer in the sample corresponding to the concentration of the analyte in the sample.
In preferred embodiments, the step of determining the degree of fluorescence resonance energy transfer in the sample includes measuring light emitted by the acceptor fluorescent protein moiety. In other preferred embodiments, determining the degree of fluorescence resonance energy transfer in the sample includes measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety. In yet other preferred embodiments, determining the degree of fluorescence resonance energy transfer in the sample includes measuring the excited state lifetime of the donor moiety.
The method can further include the steps of determining the concentration of the analyte at a first time after contacting the sample with the fluorescence indicator, determining the concentration of the analyte at a second time after contacting the sample with the fluorescence indicator, and calculating the difference in the concentration of the analyte at the first time and the second time, whereby the difference in the concentration of the analyte in the sample reflects a change in concentration of the analyte present in the sample.
In other embodiments, the method can further include the step of contacting the sample with a
Miyawaki Atsushi
Tsien Roger Y.
Gray Cary Ware & Friedenrich LLP
Haile Lisa A.
Parkin Jeffrey S.
Scheiner Laurie
The Regents of the University of California
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