U.S. patent application number 11/837180 was filed with the patent office on 2008-09-04 for sensor proteins and assay methods.
This patent application is currently assigned to Invitrogen Corporation. Invention is credited to Rhonda Newman, Steven Riddle, Kurt Vogel.
Application Number | 20080213811 11/837180 |
Document ID | / |
Family ID | 39733348 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080213811 |
Kind Code |
A1 |
Vogel; Kurt ; et
al. |
September 4, 2008 |
SENSOR PROTEINS AND ASSAY METHODS
Abstract
The present invention relates to biosensors. In some
embodiments, the biosensors are modified ligand binding molecules.
In some embodiments, the modified ligand binding molecule is a
phosphate binding protein (PBP). In some embodiments, the modified
ligand binding molecules are labeled to be capable of RET, e.g.,
comprising a donor and acceptor moiety. In some embodiments of the
invention, there is a detectable change in RET (e.g., FRET) when
the modified ligand binding molecule binds and/or releases the
ligand (e.g., phosphate). The invention also provides related
methods, reactions and assays.
Inventors: |
Vogel; Kurt; (Madison,
WI) ; Newman; Rhonda; (Eugene, OR) ; Riddle;
Steven; (Madison, WI) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Invitrogen Corporation
Carlsbad
CA
|
Family ID: |
39733348 |
Appl. No.: |
11/837180 |
Filed: |
August 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60822206 |
Aug 11, 2006 |
|
|
|
Current U.S.
Class: |
435/15 ; 435/19;
436/63 |
Current CPC
Class: |
Y10T 436/16 20150115;
G01N 33/542 20130101; G01N 33/84 20130101 |
Class at
Publication: |
435/15 ; 436/63;
435/19 |
International
Class: |
C12Q 1/44 20060101
C12Q001/44; G01N 33/48 20060101 G01N033/48; C12Q 1/48 20060101
C12Q001/48 |
Claims
1. A phosphate binding protein comprising a resonance energy
transfer (RET) pair of moieties comprised of at least one donor
moiety and at least one acceptor moiety, wherein the phosphate
binding protein is capable of binding a phosphate and wherein the
binding results in a change in RET.
2. The protein of claim 1, wherein RET increases.
3. The protein of claim 1, wherein RET decreases.
4. The protein of claim 1, wherein the phosphate is inorganic
phosphate (Pi).
5. The protein of claim 1, wherein the change in RET is caused by a
conformational change of the protein upon binding the
phosphate.
6. The protein of claim 1, wherein the change in RET is caused by a
conformational change of the protein upon releasing the
phosphate.
7. The protein of claim 1, wherein the distance between the at
least two moieties is altered upon binding the phosphate.
8. The protein of claim 1, wherein the orientation between the at
least two moieties is altered upon binding the phosphate.
9. The protein of claim 1, wherein the RET pair is capable of time
resolved RET.
10. The protein of claim 1, wherein the at least one acceptor
moiety is selected from the group consisting of a fluorescein, a
rhodamine, a GFP, a GFP derivatives, a fluorescent protein, a FITC,
a 5-carboxyfluorescein, a 6-carboxyfluorescein, a
7-hydroxycoumarin-3-carboxamide, a
6-chloro-7-hydroxycoumarin-3-carboxamide, a
fluorescein-5-isothiocyanate, a gdichlorotriazinylaminofluorescein,
a tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-6-isothiocyanate, a succinimidyl ester of
5-carboxyfluorescein, a succinimidyl ester of 6-carboxyfluorescein,
a 5-carboxytetramethylrhodamine, a 6-carboxymethylrhodamine, a
7-amino-4-methylcoumarin-3-acetic acid, Alexa Fluor 488, Alexa
Fluor 633, Alexa Fluor 647, 6-IAF, 5-IAF, BODIPY FL maleimide,
BODIPY FL iodoacetamide, fluorescein-5-maleimide, Oregon Green 488
iodoacetamide, Oregon Green 488 maleimide and
5-(bromomethyl)fluorescein.
11. The protein of claim 1, wherein the donor moiety comprises a
luminescent metal complex.
12. The protein of claim 11, wherein the luminescent metal complex
comprises an organic antenna moiety, a metal liganding moiety and a
lanthanide metal ion.
13. The protein of claim 12, wherein the luminescent metal complex
is a lanthanide metal complex.
14. The protein of claim 13, wherein the lanthanide metal complex
comprises an organic antenna moiety, a metal liganding moiety and a
lanthanide metal ion.
15. The protein of claim 14, wherein the lanthanide metal ion is
selected from the group consisting of: Sm(M), Ru(III), Eu (III),
Gd(III), Tb(III), and Dy(III).
16. The protein of claim 14, wherein the lanthanide ion is a
Europium ion.
17. The protein of claim 14, wherein the lanthanide ion is a
Terbium ion.
18. The protein of claim 14, wherein the organic antenna moiety is
selected from the group consisting of: rhodamine 560, fluorescein
575, fluorescein 590, 2-quinolone, 4-quinolone,
4-trifluoromethylcoumarin (TFC),
7-diethyl-amino-coumarin-3-carbohydrazide,
7-amino-4-methyl-2-coumarin (carbostyril 124),
7-amino-4-methyl-2-coumarin (coumarin 120),
7-amino-4-trifluoromethyl-2-coumarin (coumarin 124), and
aminomethyltrimethylpsoralen.
19. The protein of claim 14, wherein the metal liganding moiety is
a metal chelating moiety selected from the group consisting of:
EDTA, DTPA, TTHA, DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP,
DO3A, DOTAGA, and NOTA.
20. The protein of claim 13, wherein the lanthanide metal complex
has a structure: -L.sub.n-A-S.sub.n-C.sub.M, or
-L.sub.n-C.sub.M-S.sub.n-A, wherein A represents an organic antenna
moiety; L represents a linker; S represents a spacer; n can be 0 or
1; C represents a metal chelating moiety; and M represents a
lanthanide metal ion coordinated to C.
21. The protein of claim 11, wherein the luminescent metal complex
comprises CS124-DTPA-Phe-NCS-Tb or CS124-DTPA-EMCH-Th.
22. The protein of claim 1, wherein the protein has at least one
non-native cysteine amino acid.
23. The protein of claim 22, wherein the first or second moiety is
attached to the non-native cysteine amino acid.
24. The protein of claim 1, wherein the protein has at least two
non-native cysteine amino acids.
25. The protein of claim 24, wherein the first and second moieties
are attached to the non-native cysteine amino acids.
26. The protein of claim 22, wherein the at least one non-native
cysteine amino acids is introduced by substituting or inserting the
cysteine amino acid into the protein.
27. The protein of claim 1, wherein the amino acid sequence of the
PBP is derived from the phoS gene.
28. The protein of claim 27, wherein the amino acid sequence
encoded by the phoS gene is SEQ ID NO: 1 or SEQ ID NO:2.
29. The protein of claim 27, wherein the protein has at least one
non-native cysteine amino acid.
30. The protein of claim 27, comprising an amino acid substitution
selected from the group consisting of A47C, A197C, Q201C and
E268C.
31. The protein of claim 27, wherein the protein has at least two
non-native cysteine amino acids.
32. The protein of claim 31, comprising an amino acid substitution
selected from the group consisting of A47C, A197C, Q201C and
E268C.
33. The protein of claim 31, comprising at least 2 amino acid
substitutions selected from the group consisting of A197C/E268C,
A47C/A197C, A47C/E268C, Q201C/E268C, A47C/Q201C and A
197C/Q201C.
34. The protein of claim 27, wherein the first or second moiety is
attached to a non-native cysteine amino acid.
35. The protein of claim 27, wherein the first and second moieties
are attached to non-native cysteine amino acids.
36. The protein of claim 1, wherein the phosphate binding protein
comprises an amino acid sequence 90% homologous to SEQ ID NO: 1 or
SEQ ID NO:2.
37. The protein of claim 36, wherein the phosphate binding protein
comprises at least one non-native cysteine amino acid.
38. The protein of claim 1, wherein the at least one donor moiety
is linked to the phosphate binding protein via an amine or thiol
linkage.
39. The protein of claim 1, wherein the at least one acceptor
moiety is linked to the phosphate binding protein via an amine or
thiol linkage.
40. A method of measuring phosphate in a first sample comprising:
(a) contacting the first sample with a protein of claim 1; (b)
exposing (a) to a wavelength of light that excites the donor moiety
of the RET pair; and (c) measuring the emission from the acceptor
moiety of the RET pair.
41. The method of claim 40, comprising measuring the emission from
the donor moiety of the RET pair.
42. The method of claim. 41, comprising calculating a ratio between
the emission of the donor and acceptor moieties of the RET
pair.
43. The method of claim 40, further comprising: (i) contacting a
second sample with a protein of claim 1, wherein the second sample
comprises a known amount of the phosphate; (ii) exposing (i) to a
wavelength of light that excites the donor moiety of the RET pair;
and (iii) measuring the emission from the acceptor moiety of the
RET pair.
44. The method of claim 43, comprising measuring the emission from
the donor moiety of the RET pair in (ii).
45. The method of claim 44, comprising calculating a ratio between
the emission of the donor and acceptor moieties of the RET pair in
(ii).
46. The method of claim 40, further comprising: (i) separately
contacting multiple samples with a protein of claim 1, wherein the
multiple samples comprise a known amount of the phosphate; (ii)
exposing (i) to a wavelength of light that excites the donor moiety
of the RET pair; and (iii) measuring the emission from the acceptor
moiety of the RET pair in each sample.
47. The method of claim 46, wherein the amount of phosphate in the
first sample is determined by comparing the emission from the first
sample to the multiple samples.
48. The method of claim 47, comprising measuring the emission from
the donor moiety of the RET pair in (iii).
49. The method of claim 48, comprising calculating a ratio between
the emission of the donor and acceptor moieties of the RET pair in
(iii).
50. The method of claim 40, wherein measuring the emission occurs
at multiple time points.
51. A method for measuring phosphodiesterase activity of a compound
comprising: a) contacting the compound and a phosphodiesterase
substrate (e.g., cAMP), b) contacting (a) with a phosphatase
capable of removing a phosphate that is no longer part of a
phosphodiester bond on the substrate; c) contacting (b) with a
modified PBP; and d) measuring fluorescence.
52. The method of claim 51, wherein the modified PBP comprises one
fluorescent label, wherein the fluorescence of the PBP differ when
bound to phosphate as compared to when it is not bound to
phosphate.
53. A method for measuring phosphodiesterase activity of a compound
comprising: a) contacting the compound and a phosphodiesterase
substrate (e.g., cAMP), b) contacting (a) with a phosphatase
capable of removing a phosphate that is no longer part of a
phosphodiester bond on the substrate; c) contacting (b) with the
phosphate binding protein of claims 1; and d) measuring RET.
54. The method of claim 53, wherein (c) is exposed to a wavelength
or wavelengths of light that excite the donor moiety.
55. The method of claim 53, wherein (a), (b), and (c) are carried
out simultaneously.
56. The method of claim 53, wherein measuring RET is done in real
time or as kinetic measurements.
57. The method of claim 52, wherein (a), (b), (c) or any
combination thereof comprises a phosphate mop.
58. The method of claim 52, wherein (a) comprises a potential
modulator of the phosphodiesterase activity of the compound.
59. The method of claim 53, wherein RET is measured in (a), (b) or
(a) and (b).
60. The method of claim 52, further comprising control
reactions.
61. A method for measuring kinase activity of a compound
comprising: a) contacting the compound and a phosphorylation
substrate for the kinase activity, b) contacting (a) with a
phosphatase capable of removing a phosphate added by the kinase
activity of the compound; c) contacting (b) with a modified PBP
comprising a RET pair; and d) measuring RET.
62. The method of claim 61, wherein (c) is exposed to a wavelength
or wavelengths of light that excite the donor moiety.
63. The method of claim 61, wherein (a), (b), and (c) are carried
out simultaneously.
64. The method of claim 61, wherein RET is measured in real time or
as kinetic measurements.
65. The method of claim 61, wherein (a), (b), (c) or any
combination thereof comprises a phosphate mop.
66. The method of claim 61, wherein (a) comprises a potential
modulator of the kinase activity of the compound
67. The method of claim 61, wherein RET is measured in (a), (b) or
(a) and (b).
68. The method of claim 61, further comprising control
reactions.
69. A method for measuring kinase activity of a compound
comprising: a) preparing a solution comprising the compound, a
phosphorylation substrate for the kinase activity, a phosphatase
capable of removing a phosphate added by the kinase activity of the
compound, and a modified PBP comprising a RET pair; and b)
measuring RET.
70. The method of claim 69, wherein RET is measured in real time or
as kinetic measurements.
71. The method of claim 69, wherein (a) comprises a phosphate
mop.
72. The method of claim 69, further comprising control reactions.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 119(e)
of U.S. Provisional Patent Application No. 60/822,206, filed Aug.
11, 2006, the entire disclosure of which is incorporated herein by
reference.
1. FIELD OF THE INVENTION
[0002] The present invention, in part, provides compositions and
methods related to molecular detection including assays for the
detection of molecules. The invention also includes reactions,
methods and assays coupled to molecular detection assays. The
invention also provides, in part, reagents for detecting
conformational changes in a ligand binding molecule (e.g., a
protein) upon binding a molecule.
2. BACKGROUND OF THE INVENTION
[0003] Biosensors are analytical tools that can be used to measure
the presence of a molecular species in a sample by combining the
molecular recognition properties of biological macromolecules with
signal transduction mechanisms that couple ligand binding to
readily detectable physical changes. Commonly, a biosensor combines
a naturally occurring macromolecule (e.g., an enzyme or an
antibody), with the generation of a suitable physical signal
particular to the molecule in question.
[0004] Escherichia coli periplasmic binding proteins are members of
a protein superfamily (bacterial periplasmic binding proteins) that
has been shown to be suited for the engineering of biosensors
(e.g., see, U.S. Pat. Nos. 5,898,069 and 6,277,627). Bacterial
periplasmic binding proteins typically comprise two domains linked
by a hinge region (Quiocho & Ledvina, Molec. Microbiol.
20:17-25, 1996). The ligand-binding site is typically located at
the interface between the two domains. The proteins typically adopt
two conformations: a ligand-free open form, and a ligand-bound
closed form, which interconvert via a hinge-bending mechanism upon
ligand binding. This global, ligand mediated conformational change
can be exploited to couple ligand binding to changes in
fluorescence intensity by positioning single fluorophores in
locations that undergo conformational changes in concert with the
global change (e.g., Brune et al., Biochemistry 33:8262-8271, 1994;
Gilardi et al., Prot. Eng. 10:479-486, 1997; Gilardi et al., Anal.
Chem. 66:3840-3847, 1994; Marvin et al., Proc. Natl. Acad. Sci. USA
94:4366-4371, 1997, Marvin and Helling a, J. Am. Chem. Soc.
120:7-11, 1998; Tolosa et al., Anal. Biochem. 267:114-120, 1999;
Dattelbaum & Lakowicz, Anal. Biochem. 291:89-95, 2001; Marvin
& Helling a, Proc. Natl. Acad. Sci. USA 98:4955 4960, 2001;
Salins et al., Anal. Biochem. 294:19-26, 2001).
[0005] In biological systems, changes in phosphorylation state and
fluctuations in the concentration of inorganic phosphate are
associated with a number of important events. Because inorganic
phosphate (Pi) is involved in a number of important biological
processes, it is often desirable to be able to measure the
concentration of Pi and changes in such concentration in biological
systems. Phosphate assays, which measure Pi concentration, are
useful in a number of diagnostic methods, as well as in research
related to the functioning of biological systems.
[0006] A number of diseases and conditions present with elevated or
depressed levels of serum inorganic phosphate concentration.
Moreover, the major energy requirements of the body are fulfilled
by deriving energy from alterations in the phosphorylation state of
nucleotides. A large number of enzymes of importance to drug
discovery consume or produce inorganic phosphate (Pi), either
directly or through coupled reactions. These enzymes include
protein and lipid phosphatases, ATPases, drug transporters,
GTPases, phosphorylases, phosphodiesterases, and prenyl
transferases. Additional applications include monitoring of
phosphate in clinical samples and in process control within the
bioproduction industry. The current standard assay for phosphate
quantitation is an absorbance assay based on malachite green, which
is sensitive to about 5 .mu.M phosphate, and robust at .about.25
.mu.M phosphate. In addition to limited sensitivity, this assay
suffers in that it is absorbance-based, and thus is far from ideal
for high throughput screening. A third disadvantage is that
malachite green can only be used in an end-point format, precluding
kinetic analysis that could be useful for lead characterization and
optimization of small molecules. It is desirable to utilize
phosphate assays having a rapid response rate, in order to monitor
the kinetics of biological and chemical processes which involve the
production or consumption of Pi.
[0007] Phosphodiesterases cleave phosphodiesters to
phospho-monoesters. Important classes of phosphodiesterases include
those that act upon cyclic nucleotide phosphodiesters (cAMP or
cGMP). Present methods to quantitate such activity depend on
binding of either the substrate (cNMP) or product (NMP) to a
binding partner (an antibody or other reagent) which can be
detected by displacement of a fluorescent moiety from that binding
partner. Such binding partners can be expensive and can show poor
specificity between substrate and product, limiting assay
performance. Methods that depend on detecting a decrease in the
amount of substrate present are problematic in that low levels of
enzyme activity can be difficult to detect. Methods which are part
of the invention, described herein, do not suffer from these
disadvantages.
[0008] Fluorescent and radiometric methods exist to detect kinase
activity. However, very few truly "generic" methods exist that can
be applied to assay a wide range of kinases. Many kinase assays
depend on the use of antibodies that detect specific phosphorylated
residues in a substrate, or on the phosphorylation of specific
peptide substrates (that may not be optimal for the kinase being
assayed). Methods which are part of the invention, described
herein, can be used with "native" kinase substrates, do not depend
on the use of radioactivity or antibodies, and detect formation of
product rather than a decrease in substrate.
[0009] There is a broad demand for phosphate detection reagents and
assays. These include, for example, both basic and applied
biochemists, in pharmaceuticals and academia, as well as high
throughput screening facilities. One need among researchers is for
sensitive, but also kinetic assays for enzymatic activities. One
need among HTS facilities is for sensitive, robust, and
miniaturizable assays, preferably, but not necessarily, resistant
to compound interference.
[0010] Citation or discussion of a reference herein is not to be
construed as an admission that such is prior art to the present
invention.
3. SUMMARY OF THE INVENTION
[0011] The present invention relates, in part, to ligand-binding
molecules (e.g., a protein) labeled with a pair of moieties
suitable for resonance energy transfer (RET) measurements (e.g.,
time resolved fluorescence resonance energy transfer (TR-FRET),
fluorescence resonance energy transfer (FRET) and luminescence
resonance energy transfer (LRET)). A pair of moieties can be
covalently attached or non-covalently attached (e.g., via antibody
binding) to the ligand-binding molecule. In some cases, one of the
moieties is covalently attached and the other is non-covalently
attached.
[0012] The invention relates, in part, to assay methods based upon
one or more of the following principles: (a) detection of a
conformational change in a ligand binding molecule; or (b)
detection of an end product or intermediate of a reaction.
[0013] The ligand binding molecule can essentially be any molecule
that binds a ligand, undergoes a conformational change upon binding
and/or release of a ligand and can be labeled directly and or
indirectly with detectable moieties, wherein a detectable signal
changes upon binding and/or release of the ligand. Ligand binding
molecules of the invention include, but are not limited to,
proteins, peptides, nucleic acids (e.g., RNA, DNA and the like) and
polymers (e.g., natural or non-natural polymers
[0014] In some embodiments, the ligand-binding molecule is a
Periplasmic Binding Protein (such as phosphate binding protein
(PBP)), which are a family of prokaryotic proteins which bind
specific ligands, such as phosphate, iron, sulfate, glutamate, or
one of many other small molecules. These proteins typically consist
of two domains linked by a hinge region (Quiocho and Ledvina 1996).
The ligand-binding site is typically located at the interface
between the two domains. Periplasmic Binding Proteins typically
adopt two conformations: a ligand-free open form and a liganded
closed form, which interconvert via a hinge-bending mechanism upon
ligand binding. In some embodiments, the ligand-binding molecule is
a human plasma phosphate binding protein (HPBP). See, e.g., Morales
et al. Structure, Vol 14, 601-609, March 2006.
[0015] TR-FRET measurements are sensitive to the distance between
the donor and acceptor moieties, the orientation of the donor and
acceptor moieties and/or due to a change in the environment of one
of the fluorophores. For example, conformational changes in a
protein labeled with both donor and acceptor moieties can lead to a
change in TR-FRET. Such conformational changes occur in many
compounds (e.g., proteins) upon ligand binding. Acceptor moieties
can be chemically attached (e.g., to amines or thiols), genetically
encoded (e.g., fusion to a fluorescent protein), or noncovalently
attached (e.g., with an antibody, streptavidin, or other
binding-protein). Donor moieties (e.g., Tb-chelate) can be attached
to the protein chemically (e.g., via amines or thiols), genetically
encoded (e.g., fusion to a fluorescent protein) or associated
noncovalently (e.g., with a fluorescently labeled antibody,
streptavidin, or other binding-protein). A PBP could be produced
recombinantly and purified, purified from native sources, or not
purified (e.g., from a cell lysate).
[0016] In some embodiments, the present invention provides
compositions and methods for the detection and quantification of
phosphate (e.g., inorganic phosphate), iron, sulfate, glutamate,
and/or a small molecule, especially in biological solutions. In
some embodiments of the invention a ligand binding molecule is a
maltose binding protein, an arabinose binding protein, a dipeptide
binding protein, a Glu/Asp BP, a Fe(III) BP, a glucose binding
protein, a histidine binding protein, a glutamine binding protein,
a ribose binding protein, or a sulfate binding protein, e.g., see
Lorimier et al. Protein Science, 2002, 11:2655-2675. In some
embodiments, a ligand binding protein binds a monosaccharide (e.g.,
arabinose, glucose, and ribose), di- and trisaccharides of glucose
(e.g., maltose), an amino acid (e.g., glutamate/aspartate,
histidine, and glutamine), di- and tripeptides, an oxyanion (e.g.,
phosphate and sulfate), or a metal ion (e.g., Fe(III)). In some
embodiments, a ligand binding molecule binds phosphate and arsenate
but not other oxyanions. In some embodiments, a ligand binding
molecule binds glucose and galactose but not other monosaccharides.
In some embodiments of the invention, a ligand binding molecule
binds ATP, NADPH, an amino acid, or a peptide. In some embodiments,
the ligand is a peptide or protein from a prokaryote, a eukaryote,
a mammal, a primate, a human, or a mouse.
[0017] In particular embodiments, the present invention relates to
a modified phosphate binding protein and the use of such a protein
in a phosphate assay. Other embodiments of the invention provide
methods for measuring, detecting and/or monitoring ligand
binding.
[0018] One embodiment of the present invention relates to a
phosphate binding protein (PBP). In some embodiments, the PBP is
modified to be capable of resonance energy transfer (RET). In some
embodiments, there is a detectable difference between the RET of
the unbound PBPs and the phosphate bound PBPs of the invention.
Therefore, the modified PBPs of the invention are capable of acting
as biosensors for phosphate (e.g., inorganic phosphate (Pi)).
Modified PBPs of the invention can be used in various assays where
the detection of phosphate is desired. Additionally, an assay
involving phosphate (e.g., kinase reaction) can be coupled to
another assay (e.g., involving phosphatase) that results in a
change in phosphate concentration (e.g., increase or decrease),
which can then be detected by the modified PBPs of the present
invention. In one embodiment, the modified PBP is derived from an
E. coli PBP, e.g., encoded by the E. coli phoS gene.
[0019] Other embodiments of the invention relate to a modified
ligand binding molecule (e.g., a Periplasmic Binding Protein) that
is capable of being used for the detection of iron, sulfate,
glutamate, and/or a small molecule.
[0020] The present invention also provides general and specific
modifications of a PBP or other ligand binding molecule. In some
embodiments, a moiety that is a component of a RET pair (e.g.,
donor or acceptor moieties) can be attached to a ligand binding
protein molecule (e.g., PBP) via a thiol linkage to a cysteine
amino acid. In particular embodiments, at least one cysteine amino
acid can be substituted or inserted into the amino acid sequence of
the ligand binding protein molecule (e.g., PBP) to allow a
component of a RET pair to be attached to the ligand binding
protein molecule (e.g., PBP) via a thiol linkage to the inserted or
substituted cysteine amino acid. In some embodiments, both the
donor and acceptor moiety are attached to the ligand binding
protein molecule (e.g., PBP) via a cysteine or thiol linkage.
[0021] In particular embodiments, a moiety that is a component of a
RET pair (e.g., donor or acceptor moieties) can be attached to a
ligand binding protein molecule (e.g., PBP) via an amine linkage to
an amino acid (e.g., lysine) of the ligand binding protein molecule
(e.g., PBP). In some embodiments, the donor or acceptor moiety is
attached to the ligand binding protein molecule (e.g., PBP) via an
amine linkage. In particular embodiments, both the donor and
acceptor moiety are attached to the ligand binding protein molecule
(e.g., PBP) via an amine linkage. In specific embodiments, the
donor or acceptor moiety is attached to the PBP via an amine
linkage and the other moiety is attached via a thiol linkage. In
some embodiments, one of the components of a RET pair are randomly
attached to the ligand binding protein molecule (e.g., PBP), for
example via amine linkage.
[0022] One advantage of some of modified ligand binding molecules
(e.g., a modified PBP) described herein is that the means of
measurement is via RET (e.g., TR-RET or TR-FRET. These methods of
detection are generally better for high throughput screening
compared to existing assays. In general, TR-FRET assays are much
less prone to interference from library compounds. Also, these
assays can be ratiometric, which results in more precise data, and
a more robust assay.
[0023] The present invention provides sensitive phosphate assays
and enables the use of less enzyme and/or other reagents. In
addition, the present invention allows for detailed kinetic
analysis of enzymes, rather than only end point assays (e.g., as
with malachite green). For high throughput assays, some embodiments
of the invention lead to reduced costs by using more sensitive
detection methods, e.g., because of the use of less enzyme and/or
other reagents. Embodiments of the present invention also allow for
more cost-effective screening and more meaningful screening data by
eliminating or reducing compound interference. Furthermore, some
formats described herein may enable the screening of new targets
that were not deemed feasible with existing technologies.
[0024] In some embodiments, modified ligand binding molecules
(e.g., modified PBPs) and methods of the invention allow, in part,
related detection assays for phosphate, iron, sulfate, glutamate,
and/or a small molecule to be miniaturized; allow, in part, for
lower amounts of reagents to be consumed; and allow, in part, for
more robust and higher quality data due to reduced compound
interference, e.g., in the case of TR-FRET related assays.
[0025] Modified ligand binding molecules and methods of the
invention can be utilized in a variety of ways including, but not
limited to, assay development, high throughput screening (HTS),
target identification, lead optimization, bioproduction,
pre-clinical investigation, and kinetic/enzymatic analyses.
Modified ligand binding molecules utilizing TR-FRET (e.g.,
utilizing a lanthanide metal complex as the donor moiety) are, in
many instances, well suited for HTS, although non-TR-FRET versions
of the modified ligand binding molecules (e.g., a modified PBP) of
the invention can also be used for HTS. Modified ligand binding
molecules (e.g., modified PBPs) of the present invention provide,
in many instances, a simple solution to assay many different
enzymes either directly or through coupled reactions.
[0026] One embodiment of the invention provides a ligand binding
molecule (e.g., a phosphate binding protein) comprising a resonance
energy transfer (RET) pair of moieties comprised of at least one
donor moiety and at least one acceptor moiety, wherein the ligand
binding molecule is capable of binding a ligand and wherein the
binding results in a change in RET. One embodiment of the invention
provides a phosphate binding protein comprising a RET pair of
moieties comprised of at least one donor moiety and at least one
acceptor moiety, wherein the phosphate binding protein is capable
of binding a phosphate and wherein the binding results in a change
in RET. In some embodiments, the RET increases upon binding the
ligand. In some embodiments, the RET decreases upon binding the
ligand. In some embodiments, the ligand is inorganic phosphate
(Pi).
[0027] In some aspects of the invention, the change in RET is
caused by a conformational change of the protein upon binding the
phosphate. In some embodiments, the change in RET is caused by a
conformational change of the ligand binding molecule upon releasing
the ligand (e.g., phosphate). In some embodiments, the distance
between the at least two moieties is altered upon binding and/or
release the ligand (e.g., phosphate). In some embodiments, the
orientation between the at least two moieties is altered upon
binding and/or release of the ligand (e.g., phosphate).
[0028] In some aspects of the invention, the RET pair is capable of
time resolved RET. In some embodiments of the invention, the at
least one acceptor moiety is selected from the group consisting of
a fluorescein, a rhodamine, a GFP, a GFP derivatives, a fluorescent
protein, a FITC, a 5-carboxyfluorescein, a 6-carboxyfluorescein, a
7-hydroxycoumarin-3-carboxamide, a
6-chloro-7-hydroxycoumarin-3-carboxamide, a
fluorescein-5-isothiocyanate, a dichlorotriazinylaminofluorescein,
a tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-6-isothiocyanate, a succinimidyl ester of
5-carboxyfluorescein, a succinimidyl ester of 6-carboxyfluorescein,
a 5-carboxytetramethylrhodamine, a 6-carboxymethylrhodamine, a
7-amino-4-methylcoumarin-3-acetic acid, Alexa Fluor 488, Alexa
Fluor 633, Alexa Fluor 647, 6-IAF, 5-IAF, BODIPY FL maleimide,
BODIPY FL iodoacetamide, fluorescein-5-maleimide, Oregon Green 488
iodoacetamide, Oregon Green 488 maleimide and
5-(bromomethyl)fluorescein. The acceptor moiety may also be a dye
wherein the dye comprises a xanthene, a cyanine, an indole, a
benzofuran, a coumarin, a borapolyazaindacine, or a semiconductor
nanocrystal.
[0029] In some embodiments, a donor moiety comprises a luminescent
metal complex. In some aspects of the invention, a luminescent
metal complex comprises an organic antenna moiety, a metal
liganding moiety and a lanthanide metal ion. In some embodiments,
the metal complex is a lanthanide metal complex. In some aspects of
the invention, a lanthanide metal complex comprises an organic
antenna moiety, a metal liganding moiety and a lanthanide metal
ion. In some embodiments, the lanthanide metal ion is selected from
the group consisting of: Sm(III), Ru(III), Eu(III), Gd(III),
Tb(III), and Dy(III). In some embodiments, the lanthanide ion is a
Europium ion or a Terbium ion. In some aspects of the invention,
the organic antenna moiety is selected from the group consisting
of: rhodamine 560, fluorescein 575, fluorescein 590, 2-quinolone,
4-quinolone, 4-trifluoromethylcoumarin (TFC),
7-diethyl-amino-coumarin-3-carbohydrazide,
7-amino-4-methyl-2-coumarin (carbostyril 124),
7-amino-4-methyl-2-coumarin (coumarin 120),
7-amino-4-trifluoromethyl-2-coumarin (coumarin 124), and
aminomethyltrimethylpsoralen. In some embodiments of the invention,
the metal liganding moiety is a metal chelating moiety selected
from the group consisting of: EDTA, DTPA, TTHA, DOTA, NTA, HDTA,
DTPP, EDTP, HDTP, NTP, DOTP, DO3A, DOTAGA, and NOTA. In some
aspects of the invention, the lanthanide metal complex has a
structure: -L.sub.n-A-S.sub.n-C.sub.M, or
-L.sub.n-C.sub.M-S.sub.n-A, wherein A represents an organic antenna
moiety; L represents a linker; S represents a spacer; n can be 0 or
1; C represents a metal chelating moiety; and M represents a
lanthanide metal ion coordinated to C. In some embodiments, a
luminescent metal complex comprises CS124-DTPA-Phe-NCS-Tb or
CS124-DTPA-EMCH-Tb.
[0030] In some aspects of the invention, a ligand binding molecule
(e.g., a PBP) comprises at least one non-native cysteine amino
acid. In some embodiments, a first or second moiety is attached to
a non-native cysteine amino acid. In some embodiments of the
invention, the protein comprises at least two non-native cysteine
amino acids. In some embodiments, a first and second moieties are
attached to the non-native cysteine amino acids. In some aspects of
the invention, at least one non-native cysteine amino acid is
introduced by substituting or inserting the at least one cysteine
amino acid into a ligand binding molecule.
[0031] In some aspects of the invention, the ligand binding
molecule is derived from and/or comprises a PBP. In some
embodiments, the amino acid sequence of the PBP is derived from the
phoS gene. In some embodiments, the amino acid sequence encoded by
the phoS gene is SEQ ID NO:1 or SEQ ID NO:2. In some aspects of the
invention, the phosphate binding protein comprises an amino acid
sequence 90% homologous to SEQ ID NO: 1 or SEQ ID NO:2. In some
embodiments of the invention, the ligand binding molecule derived
from and/or comprising a PBP further comprises at least one
non-native cysteine amino acid. In some aspects of the invention, a
PBP comprises an amino acid substitution selected from the group
consisting of A47C, A197C, Q201C and E268C. In some embodiments,
the PBP comprises at least two non-native cysteine amino acids. In
some embodiments, the PBP comprising at least two non-native
cysteine amino acids comprises an amino acid substitution selected
from the group consisting of A47C, A197C, Q201C and E268C. In some
aspects of the invention, a PBP comprises at least 2 amino acid
substitutions selected from the group consisting of A197C/E268C,
A47C/A197C, A47C/E268C, Q201C/E268C, A47C/Q201C and A197C/Q201C. In
some embodiments, a first and/or second moiety is attached to a
non-native cysteine amino acid.
[0032] In some aspects of the invention, an at least one donor
moiety and/or acceptor moiety is linked to the phosphate binding
protein via an amine or thiol linkage.
[0033] The invention also provides a method of measuring phosphate
in a first sample comprising: (a) contacting the first sample with
a ligand binding molecule of the invention comprising at least one
donor moiety and at least one acceptor moiety that are capable of
RET; (b) exposing (a) to a wavelength of light that excites the
donor moiety of the RET pair; and (c) measuring the emission from
the acceptor moiety of the RET pair and/or measuring the emission
from the donor moiety of the RET pair. In some aspects, the method
comprises calculating a ratio between the emission of the donor and
acceptor moieties of the RET pair. In some embodiments of the
invention, the method further comprises: (i) contacting a second
sample with a ligand binding molecule of the invention comprising
at least one donor moiety and at least one acceptor moiety that are
capable of RET, wherein the second sample comprises a known amount
of the ligand (e.g., phosphate); (ii) exposing (i) to a wavelength
of light that excites the donor moiety of the RET pair; and (iii)
measuring the emission from the acceptor moiety of the RET pair
and/or measuring the emission from the donor moiety of the RET pair
in (ii). In some embodiments, the method comprises calculating a
ratio between the emission of the donor and acceptor moieties of
the RET pair in (ii). In some embodiments, the method comprises:
(i) separately contacting multiple samples with a ligand binding
molecule of the invention (e.g., a PBP), wherein the multiple
samples comprise a known amount of the ligand (e.g., phosphate);
(ii) exposing (i) to a wavelength of light that excites the donor
moiety of the RET pair; and (iii) measuring the emission from the
acceptor moiety of the RET pair in each sample and/or measuring the
emission from the donor moiety of the RET pair in (iii). In some
embodiments, the method comprises calculating a ratio between the
emission of the donor and acceptor moieties of the RET pair in
(iii). The methods of the invention also provide measuring the
emission at multiple time points. In some embodiments of the
invention, the amount of ligand (e.g., phosphate) in the first
sample is determined by comparing the emission from the samples,
e.g., comparing the emission from the first sample to the multiple
samples.
[0034] In some embodiments, the donor moiety is not a GFP. In some
embodiments, the donor moiety is not an aequorin protein. In some
embodiments, the donor moiety is not a protein. In some
embodiments, the acceptor moiety is not a GFP. In some embodiments,
the acceptor moiety is not an aequorin protein. In some
embodiments, the acceptor moiety is not a protein.
4. BRIEF DESCRIPTION OF THE FIGURES
[0035] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments on the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0036] FIG. 1A shows an example of a phosphodiesterase assay and
how it can be coupled to a phosphate detection assay.
[0037] FIG. 1B shows an example of a kinase assay and how it can be
coupled to a phosphate detection assay.
[0038] FIG. 2 is a graphical representation of a ligand binding
molecule in a bound and unbound state. A ligand binding molecule is
labeled with a RET pair of moieties and binding and/or release of
the ligand causes a change in RET.
[0039] FIG. 3 represents a structure of a lanthanide metal chelate
comprising an organic antenna moiety and shows the transfer of
energy from the organic antenna moiety to the lanthanide metal
ion.
[0040] FIG. 4 shows a response of OG-PBP-Tb and Rd-PBP-Tb to
phosphate.
[0041] FIG. 5 shows a titration of phosphate against double mutant
phosphate sensors.
[0042] FIG. 6 shows detection of phosphatase activity with double
mutant sensors.
[0043] FIG. 7 shows the level of interference, if any, from various
phosphate esters using a coumarin-PBP sensor. Experimental
procedures are described in Example 6.1.
[0044] FIG. 8 shows cleavage of the phosphate esters in the
presence of phosphatase, and insensitivity of the phosphodiesters
(cAMP, cGMP) to phosphatase. Experimental procedures are described
in Example 6.2.
[0045] FIG. 9 shows results where alkaline phosphatase is titrated
against the same set of phosphate mono- and di-esters (less PNP),
as well as ATP, and fluorescence intensity is measured over the
course at 5 minutes (FIG. 9A) or one hour (FIG. 9B). Experimental
procedures are described in Example 6.3.
[0046] FIG. 10 shows a titration of calmodulin-sensitive
phosphodiesterase from bovine brain that is incubated with 100 uM
cAMP (FIG. 10A) or cGMP (FIG. 10B) in the presence of 1 uM
coumarin-PBP sensor, 10 uM Ca.sup.2+, with or without 1 unit/mL
alkaline phosphatase and 1 unit/mL calmodulin. Experimental
procedures are described in Example 6.4.
[0047] FIG. 11 shows an example of a phosphodiesterase assay
coupled reaction performed in "kinetic mode". Experimental
procedures are described in Example 6.4
[0048] FIG. 12 is one representation of the present invention.
LBD=Ligand binding domain. M1 and M2 represent moiety 1 and moiety
2 respectively. FIG. 12A depicts both of the moieties are directly
attached to the ligand binding molecule. FIG. 12B depicts M1 as
bound to the ligand binding molecule via a binding partner (e.g.,
an antibody). Another embodiment of the invention, not shown here
provides both of the moieties being bound via binding partners.
[0049] FIG. 13 shows results of an assay with Fl-PBP-Tb and
measuring emission at 520 nm. Experimental procedures are described
in Example 9.
[0050] FIG. 14 shows results of an assay with Fl-PBP-Tb and
measuring the ratio of emission at 520 nm to 615 nm. Experimental
procedures are described in Example 9.
[0051] FIG. 15 shows results of an assay with Fl-PBP-Tb and
measuring the ratio of emission at 520 nm to 615 nm. Experimental
procedures are described in Example 9.
5. BRIEF DESCRIPTION OF THE SEQUENCES
[0052] SEQ ID NO:1 is an amino acid sequence of an E. coli
phosphate binding protein (phoS). This sequence includes a signal
sequence, which is underlined. (GenBank Accession No. AAA24378)
TABLE-US-00001 MKVMRTTVATVVAATLSMSAFSVFAEASLTGAGATFPAPVYAKWADTYQK
ETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLSDEKLAQEGLFQFPTV
IGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKL
PSQNIAVVRRADGSGTSFVFTSYLAKVNEEWKNNVGTGSTVKWPIGLGGK
GNDGIAAFVQRLPGAIGYVEYAYAKQNNLAYTKLISADGKPVSPTEENFA
NAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGTEVLK
FFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLY
[0053] SEQ ID NO:2 an amino acid sequence of an E. coli phosphate
binding protein (phoS), which does not include the signal
sequence.
TABLE-US-00002 EASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTV
DFGASDAPLSDEKLAQEGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTL
GDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVRRADGSGTSFVFTSYLA
KVNEEWKNNVGTGSTVKWPIGLGGKGNDGIAAFVQRLPGAIGYVEYAYAK
QNNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAW
PITSTTFILIHKDQKKPEQGTEVLKFFDWAYKTGAKQANDLDYASLPDSV
VEQVRAAWKTNIKDSSGKPLY
6. DETAILED DESCRIPTION
[0054] Generally, the nomenclature used herein and many of the
fluorescence, luminescence, computer, detection, chemistry, and
laboratory procedures described herein are commonly employed in the
art. Standard techniques are generally used for chemical synthesis,
fluorescence or luminescence monitoring and detection, optics,
molecular biology, and computer software and integration. Chemical
reactions, cell assays, and enzymatic reactions are typically
performed according to the manufacturer's specifications where
appropriate. See, generally, Lakowicz, J. R. Topics in Fluorescence
Spectroscopy, (3 volumes) New York: Plenum Press (1991), and
Lakowicz, J. R. Emerging applications of florescence spectroscopy
to cellular imaging: lifetime imaging, metal-ligand probes, multi
photon excitation and light quenching, Scanning Microsc. Suppl.
Vol. 10 (1996) pages 213-24, for fluorescence techniques; Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2ed. (1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for
molecular biology methods; Cells: A Laboratory Manual, 1st edition
(1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., for cell biology methods; and Optics Guide Melles Griot.RTM.
Irvine Calif., and Optical Waveguide Theory, Snyder & Love
(published by Chapman & Hall) for general optical methods, all
of which are incorporated herein by reference.
[0055] General methods for performing a variety of fluorescent or
luminescent assays on luminescent materials are known in the art
and are described in, e.g., Lakowicz, J. R., Topics in Fluorescence
Spectroscopy, volumes 1 to 3, New York: Plenum Press (1991);
Herman, B., Resonance Energy Transfer Microscopy, in Fluorescence
Microscopy of Living Cells in Culture, Part B, Methods in Cell
Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego
Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular
Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.
(1978), pp. 296-361; and Bernard Valeur, "Molecular Fluorescence:
Principles and Applications" Wiley VCH, 2002. Guidance in the
selection and use of specific resonance acceptor moieties is
available at, for example, Berlman, I. B., Energy transfer
parameters of aromatic compounds, Academic Press, New York and
London (1973), which contains tables of spectral overlap integrals
for the selection of resonance energy transfer pairs. Additional
information sources include the Molecular Probes Catalog (2003) and
website; Invitrogen New Products Catalog (2006); Full Invitrogen
Catalog (2005); Invitrogen Drug Discovery Solutions Catalog (2004);
Invitrogen's website; and Tsien et al., 1990 Handbook of Biological
Confocal Microscopy, pp. 169-178. Instruments useful for performing
FP and/or RET and TR-RET applications are available from Tecan
Group Ltd. (Switzerland) (Ultra, Ultra 384, Ultra Evolution);
Perkin-Elmer (Boston, Mass.) (Fusion, EnVision, Victor V, and
ViewLux), Amersham Bioscience (Piscataway, N.J.) (LeadSeeker); and
Molecular Devices Corporation (Sunnyvale, Calif.) (Analyst AD, GT,
and HT).
[0056] The term "RET" stands for resonance energy transfer, and
refers to the transmission (e.g., radiationless) of an energy
quantum from its site of absorption (the donor) to the site of its
utilization (the acceptor) in a molecule, or system of molecules,
by resonance interaction between donor and acceptor species, over
distances considerably greater than interatomic. A donor is a
moiety that initially absorbs energy (e.g., optical energy or
electronic energy). A luminescent metal complex, as described
herein, can comprise two donors: 1) an organic antenna moiety,
which absorbs optical energy (e.g., from a photon); and 2) a
lanthanide metal ion, which absorbs electronic energy (e.g.,
transferred from an organic antenna moiety). RET is sometimes
referred to as fluorescent resonance energy transfer or Forster
resonance energy transfer (both abbreviated FRET). RET includes
TR-FRET, FRET and LRET. LRET uses the term "luminescence" which is
more general than the term "fluorescence" (as in FRET).
[0057] The term "energy transfer pair" or RET pair as used herein
refers to any two moieties that participate in energy transfer. In
some embodiments, one of the moieties acts as a fluorescent
reporter, e.g., a donor, and the other acts as an acceptor, which
may be a quenching compound or a compound that absorbs and re-emits
energy in the form of a detectable signal, e.g., a fluorescent
signal ("Fluorescence resonance energy transfer." Selvin P. (1995)
Methods Enzymol 246:300-334; dos Remedios C. G. (1995) J. Struct.
Biol. 115:175-185; "Resonance energy transfer: methods and
applications." Wu P. and Brand L. (1994) Anal Biochem 218:1-13).
RET is a distance-dependent and/or orientation-dependent
interaction between two moieties in which excitation energy, (e.g.,
light) is transferred from a donor to an acceptor without emission
of a photon. Deuschle et al. (Protein Sci. 2005 14: 2304-2314) and
Smith et al. (Protein Science, 2005, 14:64-73) describes how the
distance between the RET pair and their orientation affect RET.
[0058] The acceptor may be fluorescent and emit the transferred
energy at a longer wavelength, or it may be non-fluorescent, e.g.,
serves to diminish the detectable fluorescence of the reporter
molecule (quenching). RET may be either an intermolecular or
intramolecular event, and is typically dependent on the inverse
sixth power of the separation of the donor and acceptor, making it
useful over distances comparable with the dimensions of biological
macromolecules. Thus, the spectral properties of the energy
transfer pair as a whole, change in some measurable way if the
distance and/or orientation between the moieties is altered.
Self-quenching probes incorporating fluorescent
donor-non-fluorescent acceptor combinations have been developed for
detection of proteolysis (Matayoshi, (1990) Science 247:954-958)
and nucleic acid hybridization ("Detection of Energy Transfer and
Fluorescence Quenching" Morrison, L., in Nonisotopic DNA Probe
Techniques, L. Kricka, Ed., Academic Press, San Diego, (1992) pp.
31 1-352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53; Tyagi S.
(1996) Nat. 14(8):947-8).
[0059] The term "acceptor" or "acceptor moiety" refers to a
chemical or biological moiety that accepts energy via RET. In RET
applications, acceptors may re-emit energy transferred from a donor
moiety (e.g., fluorescent or luminescent moiety), for example as
fluorescence. In some embodiments, the transfer is via RET or
TR-RET. As used herein, a donor moiety (e.g., fluorescent or
luminescent moiety) and an acceptor moiety (e.g., fluorescent
moiety) are referred to as a "RET pair." Examples of acceptors
include coumarins and related fluorophores; xanthenes such as
fluoresceins and fluorescein derivatives; fluorescent proteins such
as GFP and GFP derivatives; rhodols, rhodamines, and derivatives
thereof; resorufins; cyanines; difluoroboradiazaindacenes; and
phthalocyanines. Acceptors, including fluorescent acceptor
moieties, can also be useful as fluorescent probes in FP assays. In
most applications, the donor and acceptor dyes are different, in
which case RET can be detected by the appearance of sensitized
fluorescence of the acceptor or by quenching of donor
fluorescence.
Ligand Binding Molecules
[0060] A ligand binding molecule used in the present invention can
essentially be any molecule that binds a ligand, undergoes a
conformational change upon binding and/or release of the ligand and
can be labeled directly and or indirectly with detectable moieties,
wherein the detectable signal changes upon the binding and/or
release of the ligand. Ligand binding molecules of the invention
include proteins, peptides, and nucleic acids (e.g., RNA, DNA,
polymers (e.g., unnatural polymers) and the like).
FIGS. 2 and 12 shows examples of some general principles for
certain embodiments of the invention. In one embodiment, the ligand
binding molecule is a nucleic acid. Nucleic acids are known to bind
various molecules including, but not limited to proteins and other
nucleic acids. In many instances, binding of a nucleic acid to a
molecule causes the nucleic acid to undergo a conformational
change. Various transcription factors and nucleic acid-binding
proteins (e.g., DNA-binding proteins) can bind a nucleic acid
resulting in a conformational change. The nucleic acid can be
directly and/or indirectly labeled with detectable moieties, e.g.,
two moieties that comprise a RET pair. Many nucleic acid binding
sites are known for numerous proteins. One skilled in the art can
determine how and where to label a nucleic acid with at least two
detectable moieties in order for there to be a change in a
detectable signal upon binding and/or release of the bound
molecule. For example, a nucleic acid sequence can be isolated or
designed so that two domains of the nucleic acid bind a molecule.
The nucleic acid labeled with two members of a FRET pair can be
designed so that the two binding domains are brought within close
proximity upon binding the ligand and in turn change the proximity
of the members of the FRET pair, thereby changing detectable
FRET.
[0061] The distance between the donor and acceptor of the RET
(e.g., FRET) pair has a significant effect on the efficiency of RET
(e.g., see Berney and Danuser, Biophysical Journal, (2003)
84:3992-4010 and Jares-Erijman and Jovin, Nature Biotechnology,
(2003) 21(11):1387-1395). Although, the orientation of the donor
and acceptor in relation to each other and their surrounding
environment has been found to affect the efficiency of RET
(Deuschle et al. Protein Sci. 2005 14: 2304-2314 and Smith et al.
Protein Science, 2005, 14:64-73).
[0062] In some embodiments of the invention, the distance between
the donor and acceptor moieties when bound and/or unbound to the
ligand is between 0.1 nm and 100 nm, 1 nm and 10 nm, 1 nm and 5 nm,
5 nm and 10 nm, and 3 nm and 7 nm. In some instances, the distance
between the members of the RET pair (e.g., the acceptor and donor
moieties) is referred to as Forster distance (e.g., Ro).
[0063] In some embodiments of the invention, more than one acceptor
and/or donor moiety are attached to the ligand binding molecule. In
some embodiments, the ligand binding molecule is modified to be
capable of FRET relay (e.g., see Watrob et al. 2003, Two-step FRET
as a structural J. Am. Chem. Soc. 125: 7336-7343 and Smith et al.
Protein Science, 2005, 14:64-73). In some embodiments, the ligand
binding molecule is modified to be triply labeled, e.g., three
fluorophores. In some embodiments, the ligand modified is labeled
(e.g., covalently and/or non-covalently or a combination thereof)
with 5-iodoacetamide fluoroscein (IAF), Cy5 maleimide mono-reactive
dye and tetramethylrhodamine-5-maleimide (TMR). This triple
combination can be capable of FRET relay where excitation energy
can be transferred from IAF to Cy5 via TMR. In some embodiments,
the FRET relay will demonstrate an increase in binding or release
of a ligand by the ligand binding molecule. FRET relays can have
utility in overcoming large distances (Watrob et al. 2003) and can
provide large Stokes shifts.
[0064] Additionally, DNA binding proteins (DBP) can be modified to
include (covalently or non-covalently) two members of a FRET pair,
wherein upon DNA binding and/or release the modified DBP exhibits a
detectable change in RET. These modified DBPs can be designed using
methods and designs similar to those described herein, e.g. those
described for modified PBPs.
[0065] Labels (e.g., donor and acceptor moieties) may be attached
to ligand binding molecules by any conventional means known in the
art. For example, a label may be attached via amines, carboxyl
residues, thiol linkage on ligand binding molecules (e.g., modified
ligand binding protein) and/or by antibody binding. In one
embodiment, linkage of at least one label is via thiol groups on
cysteine residues. In some embodiments, an antibody can provide one
of the donor moieties and a ligand binding protein can provide the
other. In some embodiments, one antibody can provide one of the
donor moieties and another antibody can provide one of the acceptor
moieties.
[0066] If appropriate, natural cysteine residues in the amino acid
sequence of a ligand binding protein may be used for the attachment
of the label. However, where no suitable natural cysteine residues
are available for label attachment, cysteine residues may be
engineered into the sequence of a ligand binding protein, e.g., by
site-directed mutagenesis. Site-directed mutagenesis can be
performed by methods well known in the art. For example, a coding
sequence for a ligand binding protein is isolated and sequenced,
and oligonucleotide probes are constructed to alter (e.g., by
recombination) the codon encoding an amino acid which is desired to
be changed into a codon encoding cysteine. The mutated gene is
subsequently expressed, e.g., in a bacterial or eukaryotic
expression system, to produce the mutated protein. In some
embodiments, the label is attached to a cysteine residue on the
protein via a linkage group which is thiol-reactive. Any
thiol-reactive linkage group may be used. For example, an
iodoacetamide linker may be used. In one embodiment, the linkage
group comprises a maleimide linker.
[0067] In some embodiments, the cysteines are introduced by
inserting a cysteine amino acid next to another amino acid. In some
embodiments, the cysteines are introduced by substituting a
cysteine amino acid in place of at least one other amino acid, i.e.
substitution. Examples of specific substitutions are described
herein wherein one amino acid is substituted with a cysteine amino
acid. Embodiments of the invention also include substituting at
least one cysteine amino acid in place of several adjacent amino
acids, e.g., substituting for 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acids.
[0068] In some embodiments of the invention, RET increases upon
binding or release of the ligand by a modified ligand binding
molecule. In one embodiment, RET increases upon ligand binding. In
some embodiments, this increase will be greater than 10%, greater
than 20%, greater than 30%, greater than 40%, greater than 50%,
greater than 60%, greater than 70%, greater than 80%, greater than
90%, greater than 100%, greater than 200%, greater than 300%,
greater than 400%, greater than 750%, greater than 1000%, greater
than 2000%, greater than 3000%, or greater than 5000%. In some
embodiments, this change in RET is greater than 10% and less than
1000%, greater than 100% and less than 400%, greater than 20% and
less than 200%, greater than 200% and less than 750%, greater than
400% and less than 1000%, greater than 750% and less than 3000%, or
greater than 1000% and less than 5000%.
[0069] In one embodiment, RET decreases upon binding or release of
the ligand by a modified ligand binding molecule. In some
embodiments, this decrease will be greater than 10%, greater than
20%, greater than 30%, greater than 40%, greater than 50%, greater
than 60%, greater than 70%, greater than 80%, or greater than 90%.
In some embodiments, this change in RET is greater than 10% and
less than 90%, greater than 20% and less than 60%, greater than 50%
and less than 90%, or greater than 10% and less than 50%. In some
embodiments, at least one member of the RET pair is attached in the
vicinity of the binding cleft of a ligand binding molecule (e.g. E.
coli PBP). In one embodiment, at least one member of the RET pair
is attached within the binding cleft of a ligand binding molecule
(e.g. E. coli PBP).
[0070] In some embodiments, FRET is the type of RET detected. In
some embodiments, TR-FRET is the type of RET detected. In some
embodiments, TR-RET is the type of RET detected.
[0071] Some embodiments of the invention can produce RET (e.g.,
FRET, TR-FRET, TR-RET or LRET) by any methods, for examples see,
Berney and Danuser, Biophysical Journal, (2003) 84:3992-4010.
[0072] In one embodiment, the modified ligand binding molecule is
labeled with a terbium chelate and an acceptor moiety (e.g., a
green fluorophore). In some embodiments, the modified ligand
binding molecule contains both an acceptor moiety and a terbium
chelate (e.g., a LanthaScreen.TM. Terbium Chelate). In some
embodiments a modified ligand binding molecule of the invention
undergoes an about 10-fold change in RET ratio upon binding
phosphate. In some embodiments a modified ligand binding molecule
of the invention is sensitive at about 250 nM. In some embodiments
a modified ligand binding molecule of the invention is robust at
about 2.5 M. In some embodiments of the invention, a modified
ligand binding molecule provides at least 2, at least 3, at least
4, at least 5 or at least 10 times greater sensitivity than
Malachite Green. Some embodiments of the invention provide assays
and methods with a z' of greater than 0.5, greater than 0.6,
greater than 0.7, greater than 0.8, or greater than 0.9. In some
embodiments, the purity of the modified ligand binding molecule
used in an assay or method of the invention is greater than 90%. In
some embodiments of the invention, the modified ligand binding
molecule is not substantially purified, e.g., from a cell lysate or
clarified cell lysate.
[0073] Care may be taken to not disrupt the ligand binding region
of a ligand binding molecule (e.g., a modified ligand binding
molecule) in a way that the ligand bound versus the unbound ligand
is not detectable or in a way that binding and/or releasing of a
ligand by the ligand binding molecule (e.g., a modified ligand
binding molecule) is abolished. In some embodiments, the ligand
binding protein is modified to alter the binding specificity and/or
binding affinity.
[0074] In particular embodiments, the ligand binding molecule is a
fragment of a ligand binding protein, e.g., a fragment of a PBP.
Many ligand proteins have been characterized (e.g., using
crystallography studies) and the binding regions of the protein are
known and characterized. Therefore, fragments of the ligand binding
protein can be determined and produced which are capable of binding
a ligand. In some embodiments of the invention, these ligand
binding fragments of the ligand binding protein can be utilized as
a modified ligand binding molecule as described herein.
[0075] In some embodiments, the ligand binding molecule does not
bind phosphate. In some embodiments, the ligand binding molecule
does not bind maltose.
Phosphate Binding Proteins
[0076] A number of proteins are known which specifically bind to
phosphate (e.g., Pi). For example, transport of phosphate into and
out of cells and organelles is executed by specific transport
proteins. In bacterial cells, it is achieved by way of a high
affinity transport system dependent on a phosphate-binding protein.
Such proteins are able to specifically recognize inorganic
phosphate, bind to it and transport it across cell membranes or
between cellular compartments. Examples of such a proteins are the
E. coli phosphate binding proteins, e.g., as encoded by the phoS
gene of E. coli. This protein is located in the periplasm of E.
coli as part of the Pi scavenging system of the bacterium, which
operates under conditions of Pi starvation. Hence, binding affinity
of this protein for Pi is high.
[0077] The phoS gene has been cloned and sequenced (Magota et al. J
Bacteriol 1984, 157(3):909-17); Surin et al, J. Bacteriol. 1984;
157(3): 772-778). Moreover, it has been determined that PBP binds
Pi tightly (Medveczky and Rosenberg, Biochem Biophys Acta. 1969
192(2):369-71) and the crystal structure of the Pi-bound form has
been solved to high resolution (Luecke and Quiocho, Nature. 1990
347(6291):402-6). These studies have shown E. coli PBP to consist
of a monomeric protein of 35 kD separated into two domains, with a
Pi-binding cleft between them. It is postulated that the Pi-binding
cleft moves between open and closed positions upon Pi binding and
release. Further examples of Pi transport proteins are reviewed by
Torriani (Bioessays 1990, 12(8):371-6). A third class of Pi-binding
proteins includes Pi-binding enzymes. Many enzymes bind Pi weakly
and some are known to bind Pi strongly. Any phosphate binding
protein can be utilized with the teachings of the present invention
including, but not limited to, those described herein.
[0078] Hirschberg et al. (Biochemistry (1996) 37:10381-10385)
present crystal structures for a A197C mutant of Escherichia coli
PBP and the same mutant labeled with
N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)-coumarin-3-carboxamide
(MDCC). Although not necessary, the crystal structure can be
utilized for designing modified PBPs of the invention.
[0079] Another phosphate binding protein is a human plasma
phosphate binding protein (HPBP). In some embodiments, the
ligand-binding molecule of the invention is a human plasma
phosphate binding protein (HPBP). See, e.g., Morales et al.
Structure, Vol 14, 601-609, March 2006.
[0080] In some embodiments, the ligand binding molecule comprises a
prokaryotic ligand binding protein or fragment thereof capable of
binding the ligand, e.g., an Escherichia coli PBP such as encoded
by a phoS gene. In some embodiments, the ligand binding molecule
comprises a eukaryotic ligand binding protein or fragment thereof
capable of binding the ligand, e.g., a HPBP such as those described
in Morales et al. Structure, Vol 14, 601-609, March 2006. In some
embodiments, the ligand binding molecule comprises a human ligand
binding protein or fragment thereof capable of binding the
ligand.
[0081] In the present application, it is shown that modification of
a phosphate binding protein for attaching thereto a donor moiety
(e.g., a Lanthanide chelate) and an acceptor moiety (e.g., a
fluorescent or luminescent label) results in a modified phosphate
binding protein which is sensitive to phosphate (e.g., Pi) and
produces a shift in luminescence characteristics when Pi is bound.
The modified protein may be used in a phosphate related assay which
is capable of following the kinetics of biological reactions
involving phosphate, as well as in conventional phosphate assays
such as clinical assays and in pollution testing. One embodiment of
the present invention provides a modified phosphate binding protein
comprising a RET pair of moieties.
[0082] In some embodiments, the phosphate binding protein is
modified in order to comprise at least one detectable label whose
detectable characteristics alter upon a change in protein
conformation which occurs on phosphate binding and/or phosphate
release. In one embodiment, the change in the detectable
characteristics is due to a conformational change upon binding or
releasing phosphate (e.g., Pi) from a modified PBP, e.g., which
changes the distance and/or orientation between a FRET pair. In one
embodiment, the distance increases. In one embodiment, the distance
decreases. In one embodiment, the orientation of the RET pair
moieties changes. In one embodiment, the environment surrounding
the donor and/or acceptor molecules changes.
[0083] A modified phosphate binding protein of the invention may be
a modified form of any phosphate binding protein. In one
embodiment, the PBP is involved in a protein from an active
transport system which transfers Pi into and out of cells and
cellular compartments.
[0084] In one embodiment, the PBP is the E. coli phoS phosphate
binding protein. PBPs can be modified as described herein, e.g.,
PBPs can be modified via native or non-native (e.g., inserted)
cysteine amino acids. In some embodiments of the invention, a
modified PBP is derived from an E. coli phoS PBP. In some
embodiments, a modified PBP is derived from SEQ ID NO: 1 or 2. In
some embodiments, a modified PBP is SEQ ID NO: 1 or 2.
[0085] Some embodiments of the invention use an E. coli phoS PBP
that comprises a native amino acid sequence. In one embodiment, the
acceptor and/or donor moieties are attached to a PBP via thioether
linkage. In one embodiment, the acceptor and/or donor moieties are
attached to a PBP via mixed disulfide linkage. In one embodiment,
the acceptor and/or donor moieties are attached to a PBP via an
antibody.
[0086] In some embodiments, the acceptor and/or donor moiety of the
RET pair is attached to the PBP via a thioester, thioether or mixed
disulfide linkage. In some embodiments, amino acid 197 (numbering
from the N-terminus of the mature PBP as shown in Magota et al (J.
Bacteriol. 1984, 157(3):909-17; e.g., SEQ ID NO:2) can be
substituted with a cysteine. An attachment site for one member of a
FRET pair can be at a cysteine residue substituted at position 197
in the amino acid sequence of a PBP, e.g., position 197 of SEQ ID
NO:2. In some embodiments, the donor or acceptor moieties are
attached to a cysteine substituted for an amino acid at a position
selected from the group consisting of 47, 154, 185, 197, 201 and
268 (numbering from the N-terminus of the mature PBP as shown in
Magota et al (J. Bacteriol. 1984, 157(3):909-17; e.g., SEQ ID
NO:2). In some embodiments, donor and/or acceptor moieties are
attached to a cysteine substituted for or added adjacent to an
amino acid, at a position selected from the group consisting of 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 180, 181, 182, 183, 184, 185, 186, 187,
188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200
201, 264, 265, 266, 267, 268, 269, 270, 271, 272, and 273
(numbering from the N-terminus of the mature PBP as shown in Magota
et al (J. Bacteriol. 1984, 157(3):909-17; e.g., SEQ ID NO:2). For
clarity, substitution includes the deletion of more than one amino
acid. For example, a deletion of amino acids 195 to 198 would be
considered a substitution of anyone of amino acids 195 to 198,
e.g., 197. In some embodiments, an amino acid is substituted with a
cysteine and the moiety is attached to the substituted cysteine,
e.g., with a thiol-reactive label. In some embodiments, at least
one cysteine is added adjacent to (e.g., inserted) one or more of
the amino acids described herein. For example, a cysteine could be
inserted adjacent to amino acid 47, 154, 185, 197, 201 or 268
(numbering from the N-terminus of the mature PBP as shown in Magota
et al (J. Bacteriol. 1984, 157(3):909-17; e.g., SEQ ID NO:2).
[0087] Various combinations for amino acid positions for attaching
the donor and acceptor moieties (numbering from the N-terminus of
the mature PBP as shown in Magota et al (J. Bacteriol. 1984,
157(3):909-17; e.g., SEQ ID NO:2) are included as embodiments of
the invention. The following are examples of "regions" where the
acceptor or donor moieties can be attached(numbering from the
N-terminus of the mature PBP as shown in Magota et al (J.
Bacteriol. 1984, 157(3):909-17; e.g., SEQ ID NO:2). Region 1
includes amino acids 42-51. Region 2 includes amino acids 149-159.
Region 3 includes amino acids 180-191. Region 4 includes amino
acids 192-201. Region 5 includes amino acids 264-273. These regions
are presented as exemplary regions and in no way is the invention
meant to be limited to these regions. In some embodiments of the
invention, at least one acceptor moiety is located in one of
regions 1 to 5 and at least one donor moiety is located in one of
the other regions of 1 to 5. In some embodiments, at least one
acceptor moiety is attached to region 1 and at least one donor
moiety is attached to region 2, 3, 4 or 5. In some embodiments, at
least one acceptor moiety is attached to region 2 and at least one
donor moiety is attached to region 1, 3, 4 or 5. In some
embodiments, at least one acceptor moiety is attached to region 3
and at least one donor moiety is attached to region 1, 2, 4 or 5.
In some embodiments, at least one acceptor moiety is attached to
region 4 and at least one donor moiety is attached to region 1, 2,
3, or 5. In some embodiments, at least one acceptor moiety is
attached to region 5 and at least one donor moiety is attached to
region 1, 2, 3, or 4.
[0088] In some embodiments, at least one donor moiety is attached
to region 1 and at least one acceptor moiety is attached to region
2, 3, 4 or 5. In some embodiments, at least one donor moiety is
attached to region 2 and at least one acceptor moiety is attached
to region 1, 3, 4 or 5. In some embodiments, at least one donor
moiety is attached to region 3 and at least one acceptor moiety is
attached to region 1, 2, 4 or 5. In some embodiments, at least one
donor moiety is attached to region 4 and at least one acceptor
moiety is attached to region 1, 2, 3, or 5. In some embodiments, at
least one donor moiety is attached to region 5 and at least one
acceptor moiety is attached to region 1, 2, 3, or 4.
[0089] In some embodiments, the acceptor and donor moieties are
attached to the same region, e.g. region 1, 2, 3, 4, or 5.
[0090] Moieties can be attached by any methods known in the art.
This includes substituting cysteines, adding cysteines or using
native cysteines to attach the moiety via thiol linkage. Moieties
may also be attached via amines. Additionally, moieties can be
attached via noncovalent means/association including, but not
limited to, antibodies, binding partners (such as biotin and
streptavidin).
[0091] Care may be taken to not disrupt the phosphate binding
region of the protein in a way that the phosphate bound versus the
unbound PBP is not detectable or in a way that binding and/or
releasing of phosphate by the PBP is abolished.
[0092] In one embodiment, RET increases upon phosphate (e.g., Pi)
binding. In some embodiments, this change in RET will be greater
than 10%, greater than 20%, greater than 30%, greater than 40%,
greater than 50%, greater than 60%, greater than 70%, greater than
80%, greater than 90%, greater than 100%, greater than 200%,
greater than 400%, greater than 750%, greater than 1000% or any
other percentages as described herein. In some embodiments, this
change in RET is greater than 10% and less than 1000%, greater than
100% and less than 400%, greater than 20% and less than 200%,
greater than 200% and less than 750%, and greater than 400% and
less than 1000%.
[0093] In one embodiment, RET decreases upon phosphate (e.g., Pi)
binding. In some embodiments, this decrease will be greater than
10%, greater than 20%, greater than 30%, greater than 40%, greater
than 50%, greater than 60%, greater than 70%, greater than 80%,
greater than 90%, or any other percentage as described herein. In
some embodiments, this change in RET is greater than 10% and less
than 90%, greater than 20% and less than 60%, greater than 50% and
less than 90%, or greater than 10% and less than 50%. In some
embodiments, at least one member of the FRET pair is attached in
the vicinity of the binding cleft of the E. coli PBP (e.g., within
the binding cleft).
[0094] In one embodiment, the modified PBP is labeled with a
terbium chelate and an acceptor moiety (e.g., a green fluorophore).
In one embodiment, the modified PBP contains both an acceptor
moiety and a terbium chelate (e.g., a LanthaScreen.TM. Terbium
Chelate). In some embodiments a modified PBP of the invention
undergoes an about 10-fold change in RET ratio upon binding
phosphate. In some embodiments a modified PBP of the invention is
sensitive at about 250 nM. In some embodiments a modified PBP of
the invention is robust at about 2.5 .mu.M. In some embodiments of
the invention, a modified PBP provides at least 2, at least 3, at
least 4, at least 5 or at least 10 times greater sensitivity than
Malachite Green. In some embodiments, a modified PBP provides 2 to
10 times greater, 3 to 5 times greater, 4 to 10 times greater
sensitivity than Malachite Green. Some embodiments of the invention
provide assays and methods with a z' of greater than 0.5, greater
than 0.6, greater than 0.7, greater than 0.8, or greater than 0.9.
Some embodiments of the invention provide assays and methods with a
z' of between 0.5 and 0.9, 0.5 to 0.7, 0.6 to 0.9, or 0.7 to
0.99.
[0095] In some embodiments, the purity of the modified PBP used in
an assay or method of the invention is greater than 90%. In some
embodiments of the invention, the modified PBP is not substantially
purified, e.g., from a cell lysate or clarified cell lysate.
Binding Partner
[0096] As used herein, binding partners refer to binding molecules
that bind the ligand binding molecule, but is not meant to include
the ligand to be detected. A "binding partner" is a compound (e.g.,
an antibody) that has affinity for another compound (e.g., a ligand
binding molecule) such that the binding partner and the compound
are capable of forming a complex when bound. For example, a first
binding partner can be a monoclonal antibody that recognizes an
epitope on the ligand binding domain. In some embodiments, the
epitope is a native epitope found on the ligand binding molecule.
In some embodiments, the epitope is introduced or engineered to the
ligand binding molecule. Epitopes that may be used include, but are
not limited to, a histidine tag (e.g., 6Xhistidine); c-myc (e.g.,
an amino acid segment of the human protooncogene myc (e.g.,
EQKLISEEDL)); haemoglutinin tag (e.g., from an influenza
hemagglutinin protein (e.g., YPYDVPDYA)); digoxigenin (a small
organic molecule that can be covalently added to proteins or
nucleic acids); and biotin.
[0097] Some embodiments of the invention utilize antibodies labeled
with a lanthanide metal complex (e.g., comprising a Tb chelate)
following standard protocols (e.g., supplied with a commercial
chelate reagent). In some embodiments, antibodies are labeled "in
situ" through association with species-specific antibodies (e.g.,
Tb-labeled anti-IgG) that bind to an anti-ligand-binding-molecule
antibody. In some embodiments, assays of the invention may be read
using standard "LanthaScreen.TM." settings, e.g., as described in
the "LanthaScreen.TM. User's Guide" (Invitrogen, California).
[0098] Accordingly, in one aspect, the invention provides
compositions that include a binding partner. A binding partner can
be labeled with a luminescent metal complex (e.g., Tb or Europium).
In some embodiments, the binding partner can be labeled with an
acceptor moiety (e.g., fluorescent). The present invention also
provides mixtures of binding partners. For example, a composition
can include a first binding partner and a second binding partner. A
first binding partner can comprise a luminescent metal complex
while the second binding partner can comprise an acceptor moiety
(e.g., fluorescent). In some embodiments, the first binding partner
can comprise a fluorescent acceptor moiety, while the second
binding partner can comprise a luminescent metal complex wherein
both binding partners will bind the ligand binding molecule.
[0099] Typically, the affinity (e.g., apparent Kd) of a first
binding partner for a ligand binding molecule is about 1 mM or
less, e.g., about 10 .mu.M or less, or about 1 mM or less, or about
0.1 .mu.M or less, or 10 nM or less, or 1 nM or less, or 0.1 nM or
less. In some embodiments, the affinity of a first binding partner
for a ligand binding molecule is between about 1 mM to 0.1 nM,
between about 10 .mu.M to 1 .mu.M, and between about 1 .mu.M to 1
nM,
[0100] As one of skill in the art will recognize, one can
systematically adjust experimental parameters, e.g., concentrations
of assay components, reaction times, temperatures, and buffers,
depending on the Kd of the binding partner for the ligand binding
molecule, to obtain a desired combination of conditions and
cost-effectiveness.
[0101] A binding partner can be a protein, polypeptide, a
polynucleotide, a lipid, a phospholipid, a polysaccharide, or an
organic molecule. Examples of specific protein or polypeptide
binding partners include an antibody, a protein, or an
enzymatically or chemically-synthesized or modified polypeptide
sequence (e.g., a polypeptide sequence derived from a protein,
modified from a protein, or designed and synthesized de novo.) A
protein or polypeptide binding partner may be linear or cyclic. An
organic molecule binding partner can be a small organic
molecule.
[0102] In some embodiments, a binding partner comprises either a
luminescent metal complex or an acceptor moiety (e.g.,
fluorescent). In some embodiments, one binding partner can comprise
a luminescent metal complex and another can comprise an acceptor
moiety (e.g., fluorescent), e.g., a first binding partner comprises
a luminescent metal complex and a second binding partner comprises
an acceptor moiety (e.g., fluorescent).
[0103] In one embodiment, an antibody can be labeled with a
luminescent metal chelate and a ligand binding molecule that the
antibody binds can be labeled with an acceptor moiety (e.g.,
fluorescent).
[0104] Binding partners can be prepared and purified by a number of
methods known to those of ordinary skill in the art. For example,
antibodies, including monoclonal antibodies and antibody fragments,
can be prepared by a number of methods known to those of skill in
the art, or can be purchased from a variety of commercial vendors,
including Serotec (Raleigh, N.C.), Abcam (Cambridge, Mass.),
R&D Systems, Cambridge Antibody Technologies, and Covance
Research Products (Denver, Colo.).
[0105] In general, an antigen for which an antibody is desired is
prepared, e.g., recombinantly, by chemical synthesis, or by
purification of a native protein, and then used to immunize
animals. For example, polypeptides or proteins containing a
particular amino acid sequence and/or post-translational
modification (e.g., phosphorylation) can be prepared by solid-phase
chemical synthesis in order to raise an antibody specific for the
sequence and/or post-translational modification. Various host
animals including, for example, rabbits, chickens, mice, guinea
pigs, goats, and rats, can be immunized by injection of the antigen
of interest. Depending on the host species, adjuvants can be used
to increase the immunological response and include Freund's
adjuvant (complete and/or incomplete), mineral gels such as
aluminum hydroxide, surface-active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are
contained in the sera of the immunized animals. Monoclonal
antibodies can be prepared using standard hybridoma technology. In
particular, monoclonal antibodies can be obtained by any technique
that provides for the production of antibody molecules by
continuous cell lines in culture as described, for example, by
Kohler et al. (1975) Nature 256:495-497, the human B-cell hybridoma
technique of Kosbor et al. (1983) Immunology Today 4:72, and Cote
et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the
EBV-hybridoma technique of Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc. pp. 77-96 (1983). Such
antibodies can be of any immunoglobulin class including IgM, IgG,
IgE, IgA, IgD, and any subclass thereof. A hybridoma producing the
monoclonal antibodies of the invention can be cultivated in vitro
or in vivo. Chimeric antibodies can be produced through standard
techniques.
[0106] Antibody fragments that have specific binding affinity for
an antigen can be generated by known techniques. Such antibody
fragments include, but are not limited to, F(ab')2 fragments that
can be produced by pepsin digestion of an antibody molecule, and
Fab fragments that can be generated by reducing the disulfide
bridges of F(ab')2 fragments. Alternatively, Fab expression
libraries can be constructed. See, for example, Huse et al. (1989)
Science 246:1275-1281. Single chain Fv antibody fragments are
formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge (e.g., 15 to 18 amino acids),
resulting in a single chain polypeptide. Single chain Fv antibody
fragments can be produced through standard techniques, such as
those disclosed in U.S. Pat. No. 4,946,778.
[0107] Once produced, antibodies or fragments thereof can be tested
for recognition of (and affinity for) a ligand binding molecule
(e.g., a PBP) by standard immunoassay methods including, for
example, enzyme-linked immunosorbent assay (ELISA) or radioimmuno
assay (RIA). See, Short Protocols in Molecular Biology, eds.
Ausubel et al., Green Publishing Associates and John Wiley &
Sons (1992). Suitable antibodies typically will have a Kd for an
antigen (e.g., a ligand binding molecule) of about 1 mM or less,
e.g., about 10 .mu.M or less, or about 1 mM or less, or about 0.1
.mu.M or less, or about 10 nM or less, or about 1 nM or less, or
about 0.1 nM or less. In some embodiments, an antibody will have a
Kd for an antigen between about 1 mM to 0.1 nM, between about 10
.mu.M to 1 .mu.M, and between about 1 .mu.M to 1 nM.
[0108] Other polypeptides in addition to antibodies are useful as
first or second binding partners and can also be prepared and
analyzed using standard methods. By way of example and not
limitation, polypeptides, proteins and antibodies can be obtained
by extraction from a natural source (e.g., from isolated cells,
tissues or bodily fluids), by expression of a recombinant nucleic
acid encoding the protein or polypeptide, or by chemical synthesis.
Polypeptides or proteins can be produced by, for example, standard
recombinant technology, using expression vectors encoding the
proteins or polypeptides. The resulting polypeptides then can be
purified. Expression systems that can be used for small or large
scale production of polypeptides include, without limitation,
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA, or
cosmid DNA expression vectors; yeast (e.g., S. cerevisiae)
transformed with recombinant yeast expression vectors; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus); plant cell systems infected with recombinant virus
expression vectors (e.g., tobacco mosaic virus) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid); or
mammalian cell systems (e.g., primary cells or immortalized cell
lines such as COS cells, Chinese hamster ovary cells, HeLa cells,
human embryonic kidney 293 cells, and 3T3 L1 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., the metallothionein promoter)
or from mammalian viruses (e.g., the adenovirus late promoter and
the cytomegalovirus promoter).
[0109] Suitable methods for purifying the polypeptides or proteins,
including antibodies or binding fragments thereof, of the invention
can include, for example, affinity chromatography,
immunoprecipitation, size exclusion chromatography, and ion
exchange chromatography. See, for example, Flohe et al. (1970)
Biochim. Biophys. Acta. 220:469-476, or Tilgmann et al. (1990) FEBS
264:95-99. The extent of purification can be measured by any
appropriate method, including but not limited to: column
chromatography, polyacrylamide gel electrophoresis, or
high-performance liquid chromatography.
[0110] Polypeptides and proteins as binding partners can also be
prepared using solid phase synthesis methods, see, e.g., PCT
Publication No. WO 03/01115 and U.S. Pat. No. 6,410,255. For ease
of synthesis and cost considerations, polypeptides synthesized
chemically can be typically between 3 to 50 amino acids (e.g., 3 to
30, 3 to 20, 3 to 15, 5 to 30, 5 to 20, 5 to 15, 8 to 20, 8 to 15,
10 to 10, 10 to 15 or 10 to 12 amino acids in length). In the
polypeptides and proteins of the invention, a great variety of
amino acids can be used. Suitable amino acids include natural,
non-natural, and modified (e.g., phosphorylated) amino acids. Amino
acids with many different protecting groups appropriate for
immediate use in the solid phase synthesis of peptides are
commercially available.
[0111] Polynucleotides useful as binding partners can be produced
by standard techniques, including, without limitation, common
molecular cloning and chemical nucleic acid synthesis techniques.
For example, polymerase chain reaction (PCR) techniques can be
used. PCR refers to a procedure or technique in which target
nucleic acids are enzymatically amplified. Sequence information
from the ends of the region of interest or beyond typically is
employed to design polynucleotide primers that are identical in
sequence to opposite strands of the template to be amplified. PCR
can be used to amplify specific sequences from DNA as well as RNA,
including sequences from total genomic DNA or total cellular RNA.
Primers are typically 14 to 40 nucleotides in length, but can range
from 10 nucleotides to hundreds of nucleotides in length. General
PCR techniques are described, for example in PCR Primer: A
Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring
Harbor Laboratory Press, 1995. When using RNA as a source of
template, reverse transcriptase can be used to synthesize
complementary DNA (cDNA) strands. Ligase chain reaction, strand
displacement amplification, self-sustained sequence replication, or
nucleic acid sequence-based amplification also can be used to
obtain isolated nucleic acids. See, for example, Lewis Genetic
Engineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl.
Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292
(1991).
[0112] Polynucleotides of the invention also can be chemically
synthesized, either as a single nucleic acid molecule (e.g., using
automated DNA synthesis in the 3' to 5' direction using
phosphoramidite technology) or as a series of smaller
polynucleotides. For example, one or more pairs of long
polynucleotides (e.g., >100 nucleotides) can be synthesized that
contain the desired sequence, with each pair containing a short
segment of complementarity (e.g., about 15 nucleotides) such that a
duplex is formed when the polynucleotide pair is annealed. DNA
polymerase is used to extend the polynucleotides, resulting in a
single, double-stranded polynucleotide.
[0113] Polynucleotides of the invention also can be obtained by
mutagenesis. For example, polynucleotides can be mutated using
standard techniques including polynucleotide-directed mutagenesis
and site-directed mutagenesis through PCR. See Short Protocols in
Molecular Biology, Chapter 8, Green Publishing Associates and John
Wiley & Sons, edited by Ausubel et al., 1992.
[0114] In some embodiments of the invention, the binding partner(s)
is utilized to bring a detectable moiety (e.g., a member of a RET
pair) in close proximity to the ligand binding molecule. In some
embodiments, the ligand binding molecule is directly labeled with
one member of a RET pair and a binding partner provides the other
member of the RET pair, e.g., see FIG. 12B. In one embodiment, two
binding partners provide both members of a RET pair to the ligand
binding molecule. In some embodiments of the invention, detectable
energy transfer or lack thereof between the RET is modulated or
changed by the release and/or binding of the ligand by the ligand
binding molecule.
[0115] Assays, methods, and modified ligand binding molecules of
the invention may utilize none, one, two or more binding
partners.
Luminescent Metal Complex
[0116] A luminescent metal complex can act as a donor fluorophore
in a RET or TR-RET assay. A luminescent metal complex is useful in
the present methods because its excited state lifetime is typically
on the order of milliseconds or hundreds of microseconds rather
than nanoseconds; a long excited state lifetime allows detection to
be monitored after the decay of background fluorescence and/or
interference from light-scattering.
[0117] Methods for covalently linking a luminescent metal complex
to a variety of molecules and proteins are known to those of skill
in the art, e.g., PCT Publication Nos. WO 96/23526; WO 01/09188, WO
01/08712, and WO 03/011115; U.S. Patent Publication No. 20050064485
and U.S. Pat. Nos. 5,639,615; 5,656,433; 5,622,821; 5,571,897;
5,534,622; 5,220,012; 5,162,508; and 4,927,923.
[0118] A luminescent metal complex includes a metal liganding
moiety, one or more lanthanide metal ions, and optionally linkers,
spacers, and organic antenna moieties.
Metal Liganding Moiety
[0119] A metal liganding moiety coordinates one or more lanthanide
metal ions to form a metal complex. Typically, a metal liganding
moiety includes one or more metal coordinating moieties X, where X
is a heteroatom electron-donating group capable of coordinating a
metal cation, such as O.sup.-, OH, NH.sub.2, OPO.sub.3.sup.2-, NHR,
or OR where R is an aliphatic group.
[0120] A metal liganding moiety can be a chelating moiety or a
cryptand moiety. If a lanthanide metal ion is coordinated to a
chelating moiety, the complex is referred to as a "metal chelate."
If a lanthanide metal ion is coordinated to a cryptand moiety, the
complex is referred to as a "metal cryptand."
[0121] A metal chelate should be stable to exchange of the
lanthanide ion. Metal chelates in most cases, but not all, will
have a formation constant (Kf) of greater than 10.sup.10 M.sup.-1.
A variety of useful chelating moieties are known to those of skill
in the art. Typical examples of chelating moieties include: EDTA,
DTPA, TTHA, DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP, DO3A,
DOTAGA, and NOTA.
[0122] In some embodiments, a luminescent metal chelate can have
the following structures:
-L.sub.n-A-S.sub.n-C.sub.M,
or
-L.sub.n-C.sub.M-S.sub.n-A,
wherein A represents an organic antenna moiety; L represents a
linker; S represents a spacer; n can be 0 or 1; C represents a
metal chelating moiety; and M represents a lanthanide metal ion
coordinated to C.
[0123] For illustrative examples of luminescent metal chelates, see
FIG. 3.
[0124] Cryptates are formed by the inclusion of a lanthanide cation
into a tridimensional organic cavity, leading to highly stable
complexes. A variety of useful cryptand moieties are known to those
of skill in the art. Examples of cryptand moieties useful in the
present methods include: trisbypyridine (TBP, e.g., TBP
pentacarboxylate), and pyridine bipyridine (e.g., pyridine
bipyridine tetracarboxylate).
[0125] Chelating and cryptand moieties can be synthesized by a
variety of methods known to those of skill in the art or may be
purchased commercially, e.g., U.S. Pat. Nos. 5,639,615; 5,656,433;
5,622,821; 5,571,897; 5,534,622; 5,220,012; 5,162,508; and
4,927,923; and PCT Publication Nos. WO 96/23526 and WO
03/011115.
Lanthanide Metal Ions
[0126] Metal liganding moieties coordinate one or more lanthanide
metal ions to form a metal complex. Lanthanide metal ions are
useful because their special electronic configuration shields the
optically active electrons, resulting in characteristic line type
emissions. As the electronic transitions of the metal ions are
forbidden by quantum mechanics rules, the emission lifetimes of
these ions are typically long (from .mu.s to msec).
[0127] Useful lanthanide metal ions include Sm(III), Ru(III), Eu
(III), Gd(III), Tb(III), and Dy(III). Methods for complexing a
metal ion to a chelating or cryptand moiety are known to those of
skill in the art, e.g., PCT Publication Nos. WO 96/23526 and WO
03/011115.
Antenna Moieties
[0128] A luminescent metal complex can optionally include an
antenna moiety (e.g., an organic antenna moiety). An organic
antenna moiety typically has a conjugated electronic structure so
that it can absorb light. The absorbed light is transferred by
intramolecular non-radiative processes from the singlet to the
triplet excited state of the antenna moiety, then from the triplet
state to the emissive level of the lanthanide ion, which then emits
characteristically long-lived luminescence (FIG. 3). It should be
noted that some metal liganding moieties can absorb light without
the inclusion of an organic antenna moiety. For example, certain
cryptand moieties that contain conjugated organic moieties, such as
tribipyridine pentacarboxylate, do not require the inclusion of a
discrete organic antenna moiety.
[0129] In some embodiments, an organic antenna moiety can be a
polynuclear heterocyclic aromatic compound. The polynuclear
heterocyclic aromatic compound can have two or more fused ring
structures. Examples of useful organic antenna moieties include
rhodamine 560, fluorescein 575, fluorescein 590, 2-quinolone,
4-quinolone, 4-trifluoromethylcoumarin (TFC),
7-diethyl-amino-coumarin-3-carbohydrazide,
7-amino-4-methyl-2-coumarin (carbostyril 124, CS124),
7-amino-4-methyl-2-coumarin (coumarin 120),
7-amino-4-trifluoromethyl-2-coumarin (coumarin 124),
CS124-DTPA-Phe-NCS-Tb (U.S Patent Publication No. US20050064485),
CS124-DTPA-EMCH-Tb (U.S Patent Publication No. US20050064485) and
aminomethyltrimethylpsoralen.
[0130] Compounds useful as organic antenna moieties can be
synthesized by methods known to those of skill in the art or
purchased commercially, e.g., U.S. Pat. Nos. 5,639,615; 5,656,433;
5,622,821; 5,571,897; 5,534,622; 5,220,012; 5,162,508; and
4,927,923.
Linkers, Spacers
[0131] Linkers and spacers can optionally be included in a
luminescent metal complex. A Linker (L) functions to link a
luminescent metal complex to a molecule. For example the linker can
attach a luminescent metal complex to a protein, e.g., to an amino
acid of the protein via a reaction with an amine or thiol, e.g.,
producing an amide, thioether, or disulfide linkage. In some
embodiments, a L can link an acetate, amine, amide, carboxylate, or
methylene functionality on a metal liganding moiety to a molecule
(e.g., a PBP). One of skill in the art can design Ls to react with
a number of functionalities on molecules (e.g., proteins or PBPs),
including, without limitation, amines, acetates, thiols, alcohols,
ethers, esters, ketones, and carboxylates. In some embodiments
where the molecule is a polypeptide, a L can cap the N-terminus,
the C-terminus, or both N- and C-termini, as an amide moiety. Other
exemplary L capping moieties include sulfonamides, ureas, thioureas
and carbamates. Ls can also include linear, branched, or cyclic
alkanes, alkenes, or alkynes, and phosphodiester moieties. The L
may be substituted with one or more functional groups, including
ketone, ester, amide, ether, carbonate, sulfonamide, or carbamate
functionalities. Specific Ls contemplated also include
NH--CO--NH--; --CO--(CH.sub.2).sub.n--NH--, where n=1 to 10;
--NH-Ph--; --NH--(CH.sub.2).sub.n--, where n=1 to 10; --CO--NH--;
--(CH.sub.2).sub.n--NH--, where n=1 to 10;
--CO--(CH.sub.2).sub.n--NH--, where n=1 to 10; and --CS--NH--.
Additional examples of Ls and synthetic methodologies for
incorporating them into metal complexes, particularly metal
complexes linked to polypeptides, are set forth in WO 01/09188, WO
01/08712, and WO 03/011115. In some embodiments, a disulfide
linkage is utilized.
[0132] A Spacer (S) can connect an organic antenna moiety to a
metal liganding moiety. In some embodiments, an S can link an
acetate, amine, or methylene functionality on a metal liganding
moiety to an organic antenna moiety. One of skill in the art can
design Ss to react with a number of functionalities on organic
antenna moieties and on metal liganding moieties, including,
without limitation, amines, acetates, thiols, alcohols, ethers,
esters, ketones, and carboxylates. Ss can include linear, branched,
or cyclic alkanes, alkenes, or alkynes, and phosphodiester
moieties. The S may be substituted with one or more functional
groups, including ketone, ester, amide, ether, carbonate,
sulfonamide, or carbamate functionalities. Specific Ss contemplated
also include NH--CO--NH--; --CO--(CH.sub.2).sub.n--NH--, where n=1
to 10; --NH-Ph--; --NH--(CH.sub.2).sub.n--, where n=1 to 10;
--CO--NH--; --(CH.sub.2).sub.n--NH--, where n=1 to 10;
--CO--(CH.sub.2).sub.n--NH--, where n=1 to 10; and --CS--NH--.
Donor and Acceptor Moieties
[0133] A modified ligand binding molecule (e.g., a modified PBP) of
the invention can include an acceptor moiety (e.g., fluorescent).
An acceptor moiety can act as an acceptor in RET or TR-RET based
assays. In general, an acceptor moiety (e.g., fluorescent) should,
but does not necessarily exhibit a good quantum yield and a large
extinction coefficient; should, but is not necessarily resistant to
collisional quenching and bleaching; and should, but is not
necessarily easily conjugated to a variety of ligand binding
molecules (e.g., PBPs) by methods known to those having ordinary
skill in the art. Suitable fluorophores include, without
limitation, fluorescein, rhodamine, FITCs (e.g.,
fluorescein-5-isothiocyanate), 5-carboxyfluorescein,
6-carboxyfluorescein, 5,6-carboxyfluorescein,
7-hydroxycoumarin-3-carboxamide,
6-chloro-7-hydroxycoumarin-3-carboxamide,
dichlorotriazinylaminofluorescein,
tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of
5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein,
5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and
7-amino-4-methylcoumarin-3-acetic acid. Other suitable fluorophores
include the Cy family of fluorophores (Cy 3, Cy3B, Cy3.5, Cy5;
available from GE Healthcare, Chalfont St. Giles, United Kingdom);
the Alexa Fluor family (available from Invitrogen, Carlsbad,
Calif.); the BODIPY family (available from Invitrogen, Carlsbad,
Calif.); carbopyronins; squarines; cyanine/indocyanines;
benzopyrylium heterocyles; and amide-bridged benzopyryliums.
[0134] Fluorescent proteins and mutants can also be used as
fluorescent acceptor moieties. Examples include firefly, bacterial,
or click beetle luciferases, aequorins, and other photoproteins
(for example as described in U.S. Pat. Nos. 5,221,623; 5,683,888;
5,674,713; 5,650,289; and 5,843,746. GFP and GFP mutants are
particularly useful in applications using Tb(III)-containing metal
complexes. A variety of mutants of GFP from Aequorea victoria have
been created that have distinct spectral properties, including
improved brightness, and enhanced expression and folding in
mammalian cells compared to the native GFP (e.g., see Table 7 of
U.S. Pat. No. 6,410,255; Green Fluorescent Proteins, Chapter 2,
pages 19 to 47, edited by Sullivan and Kay, Academic Press; and
U.S. Pat. Nos. 5,625,048; 5,777,079; and 5,804,387. Fluorescent
proteins can be attached chemically or a ligand binding protein
coding region can be engineered to express a fusion protein
comprised of a fluorescent protein. Fluorescent proteins can be
used as the acceptor moiety, donor moiety or both.
[0135] In some embodiments, an acceptor moiety (e.g., fluorescent)
for use in multiplex assays exhibits characteristics useful for
RET/TR-RET applications. For TR-RET applications, a region of the
acceptor moiety's (e.g., a fluorophore's) absorbance spectra should
overlap with a region of a donor moiety's (e.g., a luminescent
metal chelate's) emission spectra, while a region of the acceptor's
emission spectra should not overlap substantially with a region of
the donor's emission spectra. For example, regions of the emission
spectra of an organic-antenna-Tb(III)-chelate-containing metal
complex do not significantly overlap with the emission spectra for
fluorescein or rhodamine.
[0136] Examples of suitable acceptor fluorophores in TR-RET assays
using Tb(III)-containing luminescent metal complexes include:
fluorescein (and its derivatives); rhodamine (and its derivatives);
Alexa Fluors 488, 500, 514, 532, 546, 555, 568 (available from
Molecular Probes); BODIPYs FL, R6G, and TMR (available from
Molecular Probes); Cy3 and Cy3B (available from Amersham
Biosciences), and IC3 (available from Dojindo Molecular
Technologies, Gaithersburg, Md.). Examples of suitable acceptor
fluorophores in TR-RET assays using Eu(III)-containing luminescent
metal complexes include: Alexa Fluors 594, 610, 633, 647, and 660
(available from Molecular Probes); BODIPYs TR, 630/650, and 650/665
(available from Molecular Probes); Cy5 (available from Amersham
Biosciences) and IC5 (available from Dojindo Molecular
Technologies). Any fluorescent protein from any species could also
serve as a suitable acceptor e.g., wild type (native or
recombinant) or a mutant of Green Fluorescein Protein e.g., from
Aequorea Victoria, a blue fluorescent protein (BFP), a red
fluorescent protein (RFP), a cyan fluorescent protein (CFP), or a
yellow fluorescent protein (YFP). Suitable fluorophores for use in
the present invention are commercially available, e.g., from
Invitrogen, Carlsbad, Calif., Attotec (Germany), Amersham, and
Biosearch Technologies (Novato, Calif.). Methods for incorporating
fluorophores into proteins are known to those of skill in the art;
e.g., U.S. Pat. Nos. 5,898,069 and 6,410,255.
[0137] In some embodiments, the acceptor moiety is not a GFP. In
some embodiments, the acceptor moiety is not an aequorin protein.
In some embodiments, the acceptor moiety is not a protein.
[0138] In some embodiments, the donor moiety is not a GFP. In some
embodiments, the donor moiety is not an aequorin protein. In some
embodiments, the donor moiety is not a protein.
RET and TR-RET
[0139] Some embodiments of the present invention take advantage of
resonance energy transfer between a donor moiety (e.g., a
luminescent metal chelate) and an acceptor moiety (e.g.,
fluorescent moiety). In some embodiments, a donor luminescent metal
chelate is excited by light of appropriate wavelength and intensity
(e.g., within the donor antenna moiety's excitation spectrum) and
under conditions in which direct excitation of the acceptor
fluorophore is minimized. The donor moiety (e.g., a luminescent
chelate) then transfers the absorbed energy (e.g., by non-radiative
means) to the acceptor moiety (e.g., fluorescent), which
subsequently re-emits some of the absorbed energy, e.g., as
fluorescence emission at one or more characteristic wavelengths. In
TR-RET applications, the re-emitted radiation is not measured until
after a suitable delay time, e.g., 25, 50, 75, 100, 150, 200, 300,
25 to 300, to 150, 100 to 300, or 100 to 200 microseconds to allow
decay of background fluorescence, light scattering, or other
luminescence, such as that caused by the plastics used in
microtiter plates.
[0140] In some embodiments, RET can be manifested as a reduction in
the intensity of the luminescent signal from the donor moiety
(e.g., a luminescent metal complex) and/or an increase in emission
of fluorescence from the acceptor (e.g., fluorescent) moiety. In
some embodiments, the efficiency of RET is dependent on the
separation distance and/or the orientation of a luminescent metal
complex and acceptor fluorescent moiety, the luminescent quantum
yield of the donor metal ion, the spectral overlap with the
acceptor fluorescent moiety, and the extinction coefficient of the
acceptor fluorophore at the wavelengths that overlap with the
donor's emission spectra. Forster derived the relationship:
E=(F.degree.-F)/F.degree.=Ro.sup.6/(R.sup.6+Ro.sup.6)
where E is the efficiency of RET, F and F.degree. are the
fluorescence intensities of the donor in the presence and absence
of the acceptor, respectively, and R is the distance between the
donor and the acceptor. Ro, the distance at which the energy
transfer efficiency is 50% of maximum is given (in .ANG.) by:
Ro=9.79.times.10.sup.3(K.sup.2QJn.sup.-4).sup.1/6
where K.sup.2 is an orientation factor having an average value
close to 0.67 for freely mobile donors and acceptors, Q is the
quantum yield of the unquenched fluorescent donor, n is the
refractive index of the intervening medium, and J is the overlap
integral, which expresses in quantitative terms the degree of
spectral overlap. The characteristic distance Ro at which RET is
50% efficient depends on the quantum yield of the donor, the
extinction coefficient of the acceptor, the overlap between the
donor's emission spectrum and the acceptor's excitation spectrum,
and the orientation factor between the two fluorophores.
[0141] Changes in the degree of RET can be determined as a function
of a change in a ratio of the amount of luminescence/fluorescence
from the donor and acceptor moieties, a process referred to as
"ratioing." By calculating a ratio, the assay is less sensitive to,
for example, well-to-well fluctuations in substrate concentration,
photobleaching and excitation intensity, thus making the assay more
robust. This is of particular importance in automated screening
applications where the quality of the data produced is important
for its subsequent analysis and interpretation, see, e.g., U.S.
Pat. Nos. 6,410,255; 4,822,733; 5,527,684; and 6,352,672.
[0142] For example, in some embodiments of the method, a
ratiometric analysis is performed, wherein a ratio of
luminescence/fluorescent emission at two different wavelengths is
compared between a test sample and a control sample. In a typical
TR-RET-based assay, the two wavelengths can correspond to an
emission maximum for a luminescent metal complex and a fluorescent
acceptor moiety. In some embodiments, an emissions ratio of the
control sample will be about 1.5, 2, 3, 4, 5, 7, 10, 15, 20, 25,
30, 40, 50, 100, 1.5 to 100, 1.5 to 25, 10 to 40, 20 to 100, 1.5 to
100, or 50 to 100 times larger or smaller than the emissions ratio
of a test sample.
Ligand Detecting Assays
[0143] Some ligand binding molecules (e.g., PBPs) of the invention
can be used to measure the activity of any enzyme or reaction that
produces or consumes a ligand (e.g., phosphate), such as
phosphatases, phosphodiesterases (e.g., coupled reaction),
phosphorylases, ATPase, GTPases, and prenyl transferases (e.g.,
coupled reaction). Therefore, some embodiments of the invention
provide assays involving phosphatases, phosphodiesterases (e.g.,
coupled reactions/assays), phosphorylases, ATPase, GTPases, prenyl
transferases (e.g., coupled reactions/assays). In some embodiments,
modified PBPs of the invention are used to measure phosphate that
is a direct product of or directly consumed by a reaction of
interest, e.g., a phosphatase reaction. In some embodiments,
modified PBPs of the invention are used to measure phosphate that
is an indirect product of or indirectly consumed by a reaction of
interest, e.g., a kinase reaction coupled to a phosphatase reaction
(FIG. 1).
[0144] The present application describes various examples of
methods, assays and assay formats. One skilled in the art will
readily recognize other formats and assays and various permutations
based on the teaching herein. The present invention encompasses and
contemplates these embodiments. For example, PBP is used herein as
an example of a ligand binding molecule and the assays and assay
formats described herein using a PBP can be utilized for other
ligand binding molecules.
[0145] In one aspect of the invention, there is provided a method
for detecting phosphate in a sample comprising the steps of: (a)
contacting a sample with a modified ligand binding protein (e.g., a
PBP) comprising a donor and acceptor moiety of a FRET pair; (b)
exposing (a) to a wavelength of light that excites the donor
moiety; and (c) detecting energy (e.g., light) emitted from the
acceptor moiety. In one embodiment, the method further comprises
detecting both the energy (e.g., light/fluorescence) emitted from
the donor and acceptor moieties. In some embodiments, a ratiometric
calculation can be calculated between the light emitted from the
donor versus the acceptor moiety.
[0146] In some embodiments, a phosphatase assay can release a
phosphate which can then be detected and measured using a modified
PBP or other ligand binding molecule of the present invention,
thereby directly measuring and/or detecting the phosphatase
activity. Embodiments of the invention can be used in reactions
that directly liberate or consume phosphate or in reactions that
can be coupled through another reaction to generate or consume
phosphate.
[0147] In some embodiments, the assay is a coupled assay. For
example, a kinase assay can result in the phosphorylation of a
protein. Then a phosphatase can be utilized which removes the
phosphate from the phosphorylated protein and then a modified PBP
of the present invention can be utilized to detect and/or measure
the removed phosphate, thereby indirectly measuring and/or
detecting the kinase activity, e.g., see, FIG. 1B.
[0148] Other embodiments of the invention provide a coupled
phosphodiesterase assay. For example, cAMP is converted to AMP by
phosphodiesterase. Then AMP is converted to adenosine and phosphate
via an appropriate phosphatase. The phosphate is then detected
using a modified PBP of the invention, e.g., see, FIG. 1A for a
graphic representation of a coupled phosphodiesterase assay. For
example, the above described coupled phosphodiesterase assay can be
used to measure amounts or activity of a phosphodiesterase enzyme
or measure the amounts of cAMP in a sample.
[0149] Therefore, the methods and assays of the invention can be
utilized to measure or detect a variety of compounds, reactions or
events.
[0150] The invention provides, in part, methods for monitoring the
progression of a reaction. Ligand binding proteins of the present
invention can exhibit detectable changes upon binding and/or
release of the ligand. These changes can be utilized to monitor a
reaction. The reaction can involve the production or reduction of
the ligand in a sample or reaction. The concentration of the ligand
can be measured in real-time (e.g. kinetic measurements), as time
points or as end points, usually after a period of time. Ligand
binding molecules of the invention can be added directly to a
reaction where monitoring, detecting or quantitating a particular
ligand is desired. For example, a ligand binding molecule (e.g., a
PBP) of the invention can be added directly to a phosphatase
reaction (e.g. at the start of the reaction). In some embodiments,
the concentration of the ligand (e.g., phosphate) can be measured
directly in the reaction. In some embodiments, an aliquot of the
reaction is removed at a desired time point and the ligand
concentration and/or quantity is measured. In some embodiments, the
ligand binding molecule of the invention is contacted with the
reaction after the reaction has been started or even stopped and
the ligand concentration and/or quantity is measured. In some
embodiments, more than one ligand binding molecule of the invention
is utilized to monitor the concentration of one or multiple
ligands.
[0151] Ligand binding molecules of the invention allow measuring
and/or monitoring of a reaction and/or the concentration of the
ligand. Ligand binding molecules of the invention allow for
versatile types of measurements, e.g., real-time (e.g. kinetic
measurements), as time points or as end points. Typically real-time
or kinetic measurements are carried out by contacting the ligand
binding molecule of the invention with a reaction to be monitored.
For example, the ligand binding molecule is added to the reaction
mixture, e.g. at the start of the reaction. Then measurements are
conducted at desired time points directly in the reaction. Another
method is to remove aliquots from the reaction (e.g., at desired
time points) and measure the ligand concentration using a ligand
binding molecule of the invention. Using certain embodiments of the
present invention, it is possible to follow the kinetics of
biological systems due to the rapid reaction time of the
method.
[0152] In some embodiments related to coupled assays involving
phosphatase, it is desired that the phosphatase exhibits minimal
activity towards a substrate molecule, and high activity towards a
product molecule. For example, in a kinase assay the phosphatase
should exhibit minimal activity towards the substrate to be
phosphorylated and the phosphatase should exhibit higher activity
for the substrate after it is phosphorylated by the kinase. In some
embodiments, it is desired that the phosphatase shows minimal
activity towards ATP, and such phosphatases have been described in
the literature (e.g., Arch. Biochem. Biophys. (1978), 191(2),
613-624). In some embodiments of the invention, it is desired that
the phosphate sensor is insensitive to the presence of
substrate.
[0153] Some methods of the present invention include using control
reactions, e.g., to make a quantitative and/or qualitative measure
in an assay. In some embodiments, a reaction is compared to results
from previous assays and control reactions are not performed at the
same time as the sample is assayed. Control reactions include, but
are not limited to, those missing a component of the assay or
reaction and those that contain a known amount of a component (e.g.
a positive control and/or for quantitative measurements). Control
reactions include some but not all of the components of a reaction,
measuring the reaction, and then adding the rest of the components
and allowing the reaction to proceed and then measured the
reaction. For example, in an assaying that measures a ligand using
a ligand binding molecule of the invention, all of the components
can be added except for the sample containing the ligand. Then a
measurement can be recorded, e.g., as a negative control and/or
baseline. Then the ligand can be added and then (e.g., after a
period of time) a measurement is taken which relates to the amount
of ligand.
[0154] In some embodiments, a "phosphate mop" is used to reduce the
background levels of phosphate and/or to remove phosphate slowly
from the phosphate binding protein, thus leaving the binding site
free to detect phosphate (e.g., Pi) which may be released into the
assay system. This can ensure that phosphate binding by the
modified PBP is transient. Permanent binding may reduce the useful
life of an assay system as eventually all the phosphate binding
protein binding sites would become occupied (e.g., saturated). In
one embodiment, the phosphate mop is an enzymatic system. In some
embodiments, a 7-methyl guanosine and purine nucleoside
phosphorylase system is utilized. In some embodiments, a MESG
(2-amino-6-mercapto-7-methylpurine ribonucleoside) is utilized. In
some embodiments, a modified version of 7-methylguanosine-based
reaction including the addition of phosphodeoxyribomutase is
utilized.
[0155] According to another aspect of the invention, there is
provided a modified ligand binding protein (e.g., a phosphate
binding protein) according to the invention for use in the
measurement of a ligand (e.g., phosphate such as Pi) in the
diagnosis of a disease. Some embodiments of the invention may be
used to determine phosphate (e.g., Pi) or another ligand in methods
of diagnosis practiced in vitro or in vivo in a human or
animal.
[0156] Many methods of the invention involve measuring RET from a
ligand binding molecule (e.g., a modified ligand binding molecule)
and the amount of RET differs between the ligand bound and unbound
ligand binding molecule. In one embodiment of the invention, (a) a
ligand binding molecule (e.g., a modified ligand binding molecule)
is added to a solution; (b) RET is measured; (c) the test sample is
added to the solution; and (d) RET is measured. In some
embodiments, the RET measured in (b) is compared to (d) to
determine the presence, if any, of ligand in the test sample. In
some embodiments, the test sample solution of (c) is incubated for
a period of time to allow binding of the ligand binding molecule
(e.g., a modified ligand binding molecule) to any ligand in the
test sample. In some embodiments, (d) involves measuring RET at
multiple time points, e.g., a real time assay.
[0157] Some embodiments of the invention provide a method comprised
of (a) a first solution comprised of a test sample and a ligand
binding molecule (e.g., a modified ligand binding molecule); (b) at
least one control reaction comprised of the ligand binding molecule
(e.g., a modified ligand binding molecule) and a known
concentration of ligand; and (c) measuring RET in both (a) and (b).
"Known concentration" as referred to in (b) can be any
concentration including no ligand. In some embodiments, the RET
measured in (a) is compared to the RET measured in (b). In some
embodiments, the method comprises multiple control samples, each
comprising various known amounts of ligand. The concentration of
ligand in (a) can be determined by comparing the measured RET of
(a) to the measured RET from multiple control samples, e.g., by
using a standard curve determined from the multiple control
samples. In some embodiments, (c) involves measuring RET at
multiple time points, e.g., a real time assay. In some embodiments,
(a) and (b) are incubated for a period of time to allow binding of
the ligand binding molecule to any ligand in the test sample.
[0158] Many methods of the invention involve measuring RET from a
modified ligand binding molecule (e.g., a PBP) and the amount of
RET differs between the ligand (e.g., phosphate) bound and unbound
modified ligand binding molecule (e.g., a PBP). In one embodiment
of the invention, (a) the modified ligand binding molecule is added
to a solution; (b) RET is measured; (c) the test sample is added to
the solution; and (d) RET is measured. In some embodiments, the RET
measured in (b) is compared to (d) to determine the presence, if
any, of the ligand (e.g., phosphate) in the test sample. In some
embodiments, the test sample solution of (c) is incubated for a
period of time to allow binding of the modified ligand binding
molecule (e.g., a PBP) to any ligand in the test sample. In some
embodiments, (d) involves measuring RET at multiple time points,
e.g., a real time assay.
[0159] Some embodiments of the invention provide a method comprised
of (a) a first solution comprised of a test sample and a modified
ligand binding molecule (e.g., a PBP); (b) at least one control
reaction comprised of the modified ligand binding molecule (e.g., a
PBP) and a known concentration of ligand (e.g., phosphate); and (c)
measuring RET in both (a) and (b). "Known concentration" as
referred to in (b) can be any concentration including no ligand. In
some embodiments, the RET measured in (a) is compared to the RET
measured in (b). In some embodiments, the method comprises multiple
control samples, each comprising various known amounts of ligand.
The concentration of ligand (e.g., phosphate) in (a) can be
determined by comparing the measured RET of (a) to the measured RET
from multiple control samples, e.g., by using a standard curve
determined from the multiple control samples. In some embodiments,
(c) involves measuring RET at multiple time points, e.g., a real
time assay. In some embodiments, (a) and (b) are incubated for a
period of time to allow binding of the modified ligand binding
molecule (e.g., a PBP) to any ligand (e.g., phosphate) in the test
sample.
[0160] As discussed herein, ratiometric measurements may be used
for some methods/assays of the invention. In one embodiment,
ratiometric measurements are made by comparing the emission (e.g. a
particular or band of wavelengths of light) of both the donor and
acceptor moieties.
[0161] In some embodiments, only the emission of the donor moiety
is measured. As RET changes (e.g., due to phosphate binding), so
can the measurable emission from the donor moiety. Without wishing
to be limited by theoretical considerations, the detectable
emission from the donor moiety can change due to a change in RET
because if RET increases more energy is transferred from the donor
to the acceptor (e.g., quenching of the donor) and therefore less
detectable energy (e.g., light) is directly emitted from the donor.
If RET decreases, less energy is transferred from the donor to the
acceptor and therefore more detectable energy (e.g., light) is
directly emitted from the donor.
[0162] In some embodiments, a luminescent reference compound may be
added to the sample, and the analyte-specific signal may be
referenced relative to the signal from the reference compound or
from the reference compound and the sensor. In some embodiments,
the luminescent reference compound may be a luminescent terbium
chelate or cryptate. In some embodiments, the luminescent reference
compound may be a luminescent europium chelate or cryptate The
strategy of adding a reference fluorophore to a sample in order to
provide for an internal calibrator has been described in the
literature. (Astill et al., Clin. Chem. 33:1554-1557 (1987).)
[0163] Assays and methods of the invention can be comprised of
various formats. Some assays and methods of the invention can
optionally comprise "controls". In this aspect, a control can, for
example, be a sample of known characteristics or of unknown
characteristics that is used as a standard. The controls can
comprise multiple samples or be a single sample. In some
embodiments, the methods comprise multiple controls which can be
used to establish a standard curve, which can be utilized to
determine the concentration of a compound in a test sample. In some
embodiments, the control reaction(s) is performed in a separate
container (e.g., a tube, well, etc.) from the reaction. In some
embodiments, the control reaction(s) is performed in the container
as a test reaction. For example, a well can be loaded with
everything except the test compound and a RET measurement is
recorded, e.g., as the negative control well. Then the test
compound is added to the well and subsequently a RET measurement is
recorded. In some embodiments, the control wells or containers are
separate from the test reactions/samples. For example, one plate
(e.g., a 96 well plate) may have several wells with various
concentrations (e.g., known concentrations) of the compound to be
detected (e.g., phosphate, phosphatase, kinase, phosphodiesterase,
etc.). Another well or set of wells may contain none of the
compound to be detected. Another well or set of wells can contain
the test reactions, e.g., that have an unknown concentration of the
compounds to be detected.
[0164] In some assays, reactions and methods of the invention the
concentration of ligand binding molecule (e.g., a modified ligand
binding molecule) or PBP (e.g., modified) is about 0.1 nM to 100
nM, about 1 nM to 10 nm, about 5 nM, about 7 nM or about 2 nM. In
some embodiments, the detection mode is TR-FRET, e.g., providing
methods to detect phosphate in high throughput screens. In some
embodiments, the methods of the invention comprise assays to
determine a test compounds effect on a phosphate based assay, e.g.,
as described herein. These assays may directly produce or decrease
phosphate (e.g., Pi) or may be coupled to an assay that produces or
decreases phosphate levels.
[0165] The modified ligand binding molecules (e.g., a PBPs) of the
present invention also provide reagents, methods and assays for
identifying modulators of ligand (e.g., phosphate) related
reactions. The modified ligand binding molecules (e.g., PBPs) of
the present invention also provide reagents, methods and assays for
measuring the modulation and/or changes in ligand (e.g., phosphate)
related reactions. Phosphate related reactions include, but are not
limited to, those involving a phosphatase, a kinase, a
phosphodiesterase, prenyl transferase, phosphorylase and any
reaction that produces, removes or consumes phosphate (e.g.,
Pi).
[0166] One embodiment of the invention provides a method for
measuring kinase activity of a compound or sample comprising: a)
contacting the compound or sample and a phosphorylation substrate
for the kinase activity, b) contacting (a) with a phosphatase
capable of removing a phosphate potentially added by the kinase
activity of the compound or sample; c) contacting (b) with a
modified PBP comprising a RET pair; and d) measuring RET. In some
embodiments, (c) is exposed to a wavelength or wavelengths of light
that excite the donor moiety. In some embodiments, (a), (b), and
(c) are carried out simultaneously. For example, one solution
comprising the compound, the phosphorylation substrate, the
phosphatase capable of dephosphorylating the phosphorylated
substrate and the modified PBP is utilized. This solution can be
allowed to incubate for a period of time and then RET is measured.
In another embodiment, RET is measured in real time or as kinetic
measurements. In some embodiments, (a), (b), (c) or any combination
thereof includes a phosphate mop. In some embodiments, a potential
modulator of the kinase activity of the compound can be included in
(a). Therefore, potential modulators of kinase activity can be
screened or determined utilizing the methods of the invention. In
some embodiments, RET is measured in (a) and/or (b). Additionally,
control reactions can be set-up in the same format. Control
reactions will typically have a known amount of a particular
component involved in the reaction.
[0167] One embodiment of the invention provides a method for
measuring kinase activity of a compound or sample comprising: a) a
solution comprising the compound or sample, a phosphorylation
substrate for the kinase activity, a phosphatase capable of
removing a phosphate potentially added by the kinase activity of
the compound or sample, and a modified PBP comprising a RET pair;
and b) measuring RET. This solution can be allowed to incubate for
a period of time and then RET is measured. In another embodiment,
RET is measured in real time or as kinetic measurements. In some
embodiments, (a) includes a phosphate mop. In some embodiments, a
potential modulator of the kinase activity of the compound can be
included in (a). Therefore, potential modulators of kinase activity
can be screened or determined utilizing the methods of the
invention. Additionally, control reactions can be set-up in the
same format. Control reactions will typically have a known amount
of a particular component involved in the reaction.
[0168] A phosphodiesterase (PDE) is an enzyme that catalyzes the
hydrolysis of phosphodiester bonds. PDEs are responsible for the
degradation of the cyclic nucleotides cAMP and cGMP. They are
therefore important regulators of signal transduction mediated by
these molecules. Inhibitors of PDE can prolong or enhance the
effects of physiological processes mediated by these cyclic
nucleotides.
[0169] One embodiment of the invention provides a method for
measuring phosphodiesterase activity of a compound or sample
comprising: a) contacting the compound or sample and a
phosphodiesterase substrate (e.g., cAMP), b) contacting (a) with a
phosphatase capable of removing a phosphate that is no longer part
of a phosphodiester bond on the substrate; c) contacting (b) with a
modified PBP comprising a RET pair; and d) measuring RET. In some
embodiments, (c) is exposed to a wavelength or wavelengths of light
that excite the donor moiety. In some embodiments, (a), (b), and
(c) are carried out simultaneously. For example, one solution
comprising the compound or sample, the phosphodiesterase substrate,
the phosphatase and the modified PBP is utilized. In one
embodiment, this solution can be allowed to incubate for a period
of time and then RET is measured. In another embodiment, RET is
measured in real time or as kinetic measurements. In some
embodiments, (a), (b), (c) or any combination thereof includes a
phosphate mop. In some embodiments, a potential modulator of the
phosphodiesterase activity of the compound can be included in (a).
Therefore, potential modulators of phosphodiesterase activity can
be screened or determined utilizing the methods of the invention.
In some embodiments, RET is measured in (a) and/or (b).
Additionally, control reactions can be set-up in the same format.
Control reactions will typically have a known amount of a
particular component involved in the reaction.
[0170] The invention also provides methods to measure and/or detect
competitive binding of a compound with a ligand binding molecule
and a ligand. For example as described in, a modified ligand
binding molecule can be used to detect binding to a ligand. The
assays and methods of the invention can further include a compound
and the compound can be determined whether it is a modulator of
binding of the ligand to the ligand binding molecule. For example,
it may be desirable to determine if a compound modulates (e.g.,
inhibits or increases) binding of a ligand to a ligand binding
molecule. The compound can be added to a reaction comprising a
ligand binding molecule of the invention and a ligand. In some
embodiments, modulation is detected by comparing the RET of a
reaction with the compound to one without. In some embodiments,
differences in RET can be used to determine if the compound is a
modulator. Multiple compounds can be tested at one time, e.g., in
one reaction or in separate reactions for each compound.
Additionally, the invention provides methods and assays for
screening (e.g., libraries of) compounds for modulating binding of
a ligand for a ligand binding molecule. Additionally, the invention
provides methods and assays for screening (e.g., libraries of)
compounds that bind a ligand binding molecule. In other words, not
necessarily determining if the compound modulates binding of a
ligand to the ligand binding molecule, but determining if the
compound itself is capable of binding the ligand binding molecule,
e.g., in a similar way as a known ligand.
[0171] One embodiment of the invention provides a method for
measuring phosphodiesterase activity of a compound or sample
comprising: a) contacting the compound or sample, a
phosphodiesterase substrate (e.g., cAMP), a phosphatase capable of
removing a phosphate that is no longer part of a phosphodiesters
bond on the substrate; and a modified PBP comprising a RET pair;
and b) measuring RET. This solution can be allowed to incubate for
a period of time and then RET is measured. In another embodiment,
RET is measured in real time or as kinetic measurements. In some
embodiments, (a) includes a phosphate mop. In some embodiments, a
potential modulator of the phosphodiesterase activity of the
compound can be included in (a). Therefore, potential modulators of
phosphodiesterase activity can be screened or determined utilizing
the methods of the invention. Additionally, control reactions can
be set-up in the same format. Control reactions will typically have
a known amount of a particular component involved in the
reaction.
[0172] In part, this section describes PBPs as an example of a
phosphate binding molecule and ligand binding molecule. The
invention is not intended to be limited to PBPs as the only ligand
binding molecules that are compatible with some embodiments of the
present invention. Any phosphate or ligand binding molecule can be
used that undergoes a conformational change upon binding and/or
release of ligand and can be labeled directly and or indirectly
with detectable moieties, wherein the detectable signal changes
upon the binding and/or release of the ligand.
[0173] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
7. EXAMPLES
[0174] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples but rather should be construed
to encompass any and all variations which become evident as a
result of the teachings provided herein.
[0175] Whereas, particular embodiments of the invention have been
described herein for purposes of description, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
Example 1
Production of the Modified PBPs OG-PBP-Tb and Rd-PBP-Tb
[0176] Phosphate Binding Protein (PBP) mutant A197C can be produced
as described (Brune et al. (1994) Biochemistry 33, 8262-8271; Brune
et al. (1998) Biochemistry 37, 10370-10380). Two uL of 20 mM Oregon
Green.RTM. 488 iodoacetamide (Invitrogen, Catalog# 06010) in DMF or
20 mM Rhodamine Red.RTM. C2 maleimide (Invitrogen, Cat# R6029) in
DMSO was added to 50 uL of PBP A197C in 50 mM Tris pH 8.0 in the
presence of 200 uM 7-methylguanosine and 0.2 Units/mL Purine
Nucleoside Phosphorylase, followed by a 1 hour incubation at room
temperature. The reaction products were then desalted over a NAP-5
column (GE Healthcare) essentially according to the manufacturer's
protocol in 50 mM Tris pH 8.0 and collected in a 1 mL volume and
then dialyzed against 100 mM sodium bicarbonate pH 9.5 overnight.
The products were then concentrated in a Microcon-50 device
(Millipore) to 23 uL and 12 uL for the Oregon Green and
Rhodamine-labeled forms, respectively, and the concentration of
each was estimated using the extinction coefficient of
60,880M.sup.-1 cm.sup.-1. In one experiment, the concentrations
were found to be 166 uM and 276 uM for the Oregon Green and
Rhodamine-labeled forms, respectively.
[0177] A 3-fold molar excess of LanthaScreen.TM. Amine Reactive Tb
Chelate (Invitrogen, Cat# PV3582) was added to each from a freshly
prepared 5 mg/mL stock in 100 mM sodium bicarbonate pH 9.5. The
reactions were incubated at room temperature for 2 hours. The
reaction products were then desalted over a NAP-5 column (GE
Healthcare) essentially according to the manufacturer's protocol in
25 mM Tris pH 8.0, 1 mM MgCl.sub.2. The products of the reactions
are referred to as OG-PBP-Tb and Rd-PBP-Tb for the Oregon Green and
Rhodamine-labeled forms, respectively.
[0178] In this example, two different TR-FRET based phosphate
sensors, OG-PBP-Tb and Rd-PBP-Tb, were produced based on
fluorescently labeled forms of E. coli phosphate binding protein
(PBP), SEQ ID NO:2. They were based on the single mutant PBP A197C.
Both showed an increase in TR-FRET in response to phosphate.
Example 2
Production Mutant Sensors A47C/A197C, A197C/E268C, and Q201C
[0179] In this Example, one sensor is based on the modified PBP
A47C/A197C, a second is based on the modified PBP A197C/E268C, and
a third is based on the modified PBP Q201C.
[0180] A plasmid coding for expression of PBP A197C was modified by
site-directed mutagenesis to encode for the double mutants PBP
A47C/A197C and PBP A197C/E268C and the single mutant Q201C (Brune
et al. (1994) Biochemistry 33, 8262-8271). BL21(DE3) E. coli cells
were transformed with the plasmids and selected on LB agar plates
with tetracycline (12.5 ug/mL). 10 mL cultures of LB with
tetracycline (12.5 ug/mL) were inoculated with colonies from the
plates and grown at 37.degree. C. for 6 hours at 250 rpm. 5 mL from
these cultures was then used to inoculate 50 mL cultures of the
same media which were grown overnight at 37.degree. C. for 6 hours
at 250 rpm. 10 mL of the 50 mL cultures were used to inoculate 500
mL of TG minimal media (120 mM Tris-HCl, 80 mM NaCl, 20 mM
NH.sub.4Cl, 20 mM KCl, and 3 mM Na.sub.2SO.sub.4) with additives
(12.5 ug/mL tetracycline, 2 g/L glucose, 0.2 mM CaCl.sub.2, 10 uM
FeSO.sub.4, 0.2 mM MgSO.sub.4, and 10 mg/L thiamine) and 640 uM
KH.sub.2PO.sub.4. These 500 mL cultures were grown for
approximately 6 hours to an OD595 of approximately 2, pelleted by
centrifugation, and the cells were resuspended in the same media,
except with 64 uM KH.sub.2PO.sub.4. The cultures were then grown
for approximately 16 hours 37.degree. C. and at 250 rpm.
[0181] The cell pellets were then harvested by centrifugation and
stored at -80.degree. C., and the media is discarded. Cell pellets
from the equivalent of 500 mL of culture were suspended in 20 mL
Buffer A (20 mM Tris pH 8.2, 1 mM MgCl.sub.2) and centrifuged at
approximately 15,000.times.g for 1 hour at 4.degree. C. For PBP
A197C/E268C and PBP Q201C the supernatant was decanted and loaded
onto a 100 mL Q sepharose Fast Flow (GE Healthcare) column
equilibrated in Buffer A. The column was washed with 2 column
volumes of Buffer A and then eluted with a 2 column volume
gradient, Buffer A to Buffer B (Buffer A with 200 mM NaCl) followed
by 1 column volume of Buffer B. For PBP A47C/A197C, the supernatant
was loaded onto a 75 mL Q Sepharose column equilibrated in Buffer
A, washed with 100 mL of Buffer A, and step eluted with Buffer B.
For both proteins, the fractions were analyzed by SDS-PAGE and
fractions containing the .about.35 kDa protein are pooled.
[0182] A sample of PBP A197C/E268C was brought to 200 uL (final
concentration of 75 uM) with hepes buffered saline (HBS) (137 mM
NaCl, 2.7 mM KCl, 10 mM HEPES pH7.5) and PBP A47C/A197C was left at
25 uM in a volume of 200 uL. 1.2 uL of 50 U/mL PNPase, 2 uL of 30
mM 7-methylguanosine, and 2 uL of 100 mM MgCl.sub.2 were added to
each sample and incubated at room temperature for 45 minutes. 0.8
parts of thiol-reactive 6-iodoacetamidofluorescein (6-IAF)
(Invitrogen, Catalog# 1-30452) was added from a 10 mM stock in
DMSO. The reactions were incubated at room temperature for 5 hours
and 30 minutes and then desalted using a NAP 5 column (GE
Healthcare, Catalog# 17-0853-02) into HBS following the
manufacturer's protocol. For PBP A197C/E268C, 6.2 uL of 12 mM
LanthaScreen.TM. Thiol Reactive Tb Chelate (Invitrogen, Catalog#
PV3579) was added. For PBP A47C/A197C, 2.1 uL of 12 mM
LanthaScreen.TM. Thiol Reactive Tb Chelate was added. The Tb
labeling reactions were incubated for 1 hour at room temperature
and desalted over a NAP-5 column (GE Healthcare, Catalog#
17-0853-02) following the manufacturer's protocol. The resulting
products are referred to as Sensor(47-197) and Sensor(197-268).
Example 3
Assays with OG-PBP-Tb and Rd-PBP-Tb
[0183] The fluorescence of OG-PBP-Tb and Rd-PBP-Tb (in 25 mM Tris
pH 8.0, 1 mM MgCl.sub.2) were assessed in 22 uL reactions
containing a phosphate mop (7-methylguanosine and purine nucleoside
phosphorylase), 360 uM KH.sub.2PO.sub.4, or no additional
component. These reactions were performed at both 10 uM and 200 nM
for OG-PBP-Tb and at 9 and 0.28 uM for Rd-PBP-Tb. The samples were
excited at 340 nm (30 nm bandpass) and fluorescence intensity was
captured at 520 nm (25 nm bandpass) and 495 nm (10 nm bandpass)
using a 200 .mu.s detection window followed by a 100 .mu.s delay on
a Tecan Safire2.TM. instrument. The TR-FRET ratio was calculated by
dividing the emission intensity at 520 nm by the intensity at 495
nm.
[0184] In a set of experiments, the signals of both OG-PBP-Tb and
Rd-PBP-Tb show a strong dependence on the presence of phosphate
(FIG. 4).
Example 4
Assays with Tb-PBP(Q201C)-Fl
[0185] 10 uL of each version of Tb-PBP(Q201C)-Fl (different amount
of Tb chelate) was diluted to 100 nM with HBS and added to either
10 uL of 200 uM phosphate or to 10 uL of Phosphate Mop (0.8 mM
7-MEG, 4 U/mL PNPase, and 1 mM MgCl.sub.2). The samples were
excited at 340 nm (30 nm bandpass) and fluorescence intensity was
captured at 520 nm (25 nm bandpass) and 495 nm (10 nm bandpass)
using a 200 .mu.s detection window followed by a 100 .mu.s delay on
a Tecan Safire2.TM. instrument.
[0186] In a set of experiments, each version of Tb-PBP(Q201C)-Fl
shows a strong signal dependence on phosphate (Table 1).
TABLE-US-00003 TABLE 1 Fold-change in Emission Emission signal
(emission ratio with ratio ratio with phosphate/ Sample phosphate
with Mop ratio with Mop) Tb-PBP(Q201C)-Fl .72 .23 3.06 labeled with
3 parts Tb chelate Tb-PBP(Q201C)-Fl .78 .28 2.77 labeled with 7
parts Tb chelate Tb-PBP(Q201C)-Fl .83 .31 2.65 labeled with 10
parts Tb chelate Tb-PBP(Q201C)-Fl .85 .34 2.48 labeled with 20
parts Tb chelate
Example 5
Assays with Double Mutant Sensor(47-197) and Sensor(197-268)
[0187] The sensors were first incubated for 1 hour with 0.0039
Units/mL Purine Nucleoside Phosphorylase and 0.4 mM
7-methylguanosine, and 0.5 mM MgCl.sub.2 to remove any residual or
contaminating phosphate. 10 uL of each sensor (approximately 100
nM) was added to 10 uL of phosphate standards (final concentrations
of 1250, 625, 313, 156, 78, 39, 20, 9.8, 2.4, 1.2, 0.61, 0.31,
0.15, 0.076, 0.038, 0.019, 0.0095, 0.0048, 0.0024, 0.0012, 0.00060,
0.00030, and 0.000149 uM. The samples were excited at 340 nm (30 nm
bandpass) and fluorescence intensity was captured at 520 nm (25 nm
bandpass) and 495 nm (10 nm bandpass) using a 200 .mu.s detection
window followed by a 100 .mu.s delay on a Tecan Ultra instrument.
The TR-FRET ratio was calculated by dividing the emission intensity
at 520 nm by the intensity at 495 nm.
[0188] Results from a set of experiments are shown in FIG. 5.
Example 6
A Phosphatase Assay
[0189] PTP1B is a protein tyrosine phosphatase that negatively
regulates insulin signaling by dephosphorylating the insulin
receptor.
[0190] PTP1B (Invitrogen, Product# P3079) a recombinant human
full-length protein, histidine-tagged, expressed in insect cells)
was serially diluted across a plate in 25 mM BisTris Propane in a 5
uL volume and incubated for 30 minutes at room temperature in the
presence of 1 U/mL PNPase, 400 uM 7-methylguanosine, and 100 nM
Sensor(47-197) or Sensor(197-268). Then, 10 uL of a PTP1B substrate
(Biomol; Plymouth Meeting, Pa.; Catalog# P-323) was added to a
final concentration of 50 uM. After an approximately 1 hour
incubation, the samples were excited at 340 nm (30 nm bandpass) and
fluorescence intensity was captured at 520 nm (25 nm bandpass) and
495 nm (10 nm bandpass) using a 200 .mu.s detection window followed
by a 100 .mu.s delay on a Tecan Ultra instrument. The TR-FRET ratio
was calculated by dividing the emission intensity at 520 nm by the
intensity at 495 nm.
[0191] Results from a set of experiments are shown in FIG. 6.
Example 7
A Phosphodiesterase Assay
[0192] Some embodiments of the invention relate to the detection of
phosphodiesterase activity. Exemplary assays and their details and
results are outlined below.
6.1 Demonstration of Lack of Modified PBP Response to
Substrates.
[0193] Six different phosphate esters (phospho-serine (pSer),
phospho-threonine (pThr), phospho-tyrosine (pTyr), p-nitrophenyl
phosphate (PNP), adenosine monophosphate (AMP), and guanosine
monophosphate (GMP)), two phosphodiesters (cyclic AMP (cAMP) and
cyclic GMP (cGMP)), in addition to phosphate as a positive control,
were titrated against 1 uM coumarin labeled PBP (Invitrogen,
Carlsbad, Calif., part# PV4406) (starting at 200 uM compound
concentration), and fluorescence was measured (excite 430/5, emit
450/5).
[0194] The results from a set of experiments are shown in FIG. 7.
None of the phosphate esters or phosphodiesters showed appreciable
response in the assay.
6.2 Demonstration of Cleavage of Phosphate Esters in Presence of
Phosphatase
[0195] To demonstrate cleavage of the phosphate esters in the
presence of phosphatase, and insensitivity of the phosphodiesters
(cAMP, cGMP) to phosphatase, a large excess (8 units per 20 uL
assay well) of recombinant bovine alkaline phosphatase (Sigma, Cat#
P8361) was added to each well containing>1 uM compound and
allowed to incubate 5 minutes before measuring fluorescence
intensity as described.
[0196] In a set of experiments, all of the phosphate monoesters
showed sharp increases in fluorescence intensity, indicating full
cleavage by phosphatase. The results are shown in FIG. 8. PNP
showed a decrease in intensity at higher phosphatase concentrations
due to the fact that the product of the dephosphorylation strongly
absorbs light. cAMP and cGMP showed some increase in fluorescent
intensity, but the amount of increase was minimal relative to the
phosphate monoesters.
6.3 Determining Minimal Amount of Phosphatase to Rapidly Cleave
Phosphate Monoesters
[0197] To determine the minimal amount of phosphatase necessary to
rapidly cleave the phosphate monoesters, alkaline phosphatase was
titrated against the same set of phosphate mono- and di-esters
(less PNP), as well as ATP, and fluorescence intensity was measured
over the course of one hour.
[0198] Data from 5 minute and 1 hour time points are shown in FIGS.
9A and 9B, respectively. It can be seen that within 5 minutes, 10
milli-units/mL of alkaline phosphatase cleaved 100 mM phosphate
monoesters (or ATP) but did not cleave phosphodiesters such as cAMP
or cGMP. After one hour there was no detectible activity on cAMP or
cGMP up to about 1 unit/mL alkaline phosphatase.
6.4 Detection of Phosphodiesterase Activity Against cAMP or cGMP as
a Substrate
[0199] To demonstrate the utility of the invention in the detection
of phosphodiesterase activity against cAMP or cGMP as a substrate,
a titration of calmodulin-sensitive phosphodiesterase from bovine
brain (Sigma, Cat# P9529) was incubated with 100 uM cAMP or cGMP in
the presence of 1 uM coumarin-PBP (Invitrogen, Carlsbad, Calif.,
part# PV4406), 10 uM Ca.sup.2+, with or without 1 unit/mL alkaline
phosphatase and 1 unit/mL calmodulin (Sigma, Cat# P2277). The plate
was read (e.g., after a one hour incubation).
[0200] The data from one set of experiments using cAMP and cGMP are
shown in FIGS. 10A and 10B, respectively.
[0201] With either cAMP or cGMP as a substrate, intensity was seen
to be sensitive to the amount of phosphodiesterase present, as well
as the presence of alkaline phosphatase (an absolute requirement)
and calmodulin (the rate was increased in the presence of
calmodulin). The amount of calmodulin used in this experiment was
not optimal, and an increase in calmodulin concentration would be
expected to show a more pronounced effect.
[0202] The above experiment was also performed in "kinetic mode",
and the plate read every two minutes. When the rate of the reaction
(slope for the first .about.30% of the reaction) was plotted versus
enzyme, there was a linear relationship between rate and the
concentration of enzyme, suggesting the suitability of this format
for both HTS as well as more detailed kinetic analyses of
phosphodiesterase catalyzed hydrolysis of phosphodiesters. Results
from one experiment are shown in FIG. 11.
Example 8
Production of Fl-PBP-Tb
[0203] 268 uL of 113 uM A197C PBP was diluted with 113 uL of HBS
(137 mM NaCl, 2.7 mM KCl, 10 mM HEPES pH7.5). 15.75 .mu.L of 10 mM
6-iodoacetamidofluorescein (Invitrogen Part# 130452) in DMSO was
added. Phosphate mopping reagents were then added (1.2 .mu.L of
PNPase, 2 .mu.L of 7-MEG, and 2 .mu.L of 100 mM MgCl.sub.2) to
sequester phosphate. This reaction was incubated at room
temperature for 2.5 hours and then desalted with a NAP-5 column (GE
Healthcare) into HBS following the manufacturer's protocol. 10
.mu.L of 1 mM LanthaScreen.TM. Amine Reaction Tb Chelate
(Invitrogen Part# PV3582) in 1 M sodium bicarbonate pH 9.5 was then
added 200 .mu.L of the desalted product and incubated at room
temperature overnight. The material was then desalted with a NAP-5
column into HBS, producing Fl-PBP-Tb [Fl-PBP(A197C)-Tb(amine)].
Example 9
Assays with Fl-PBP-Tb
[0204] Add 10 .mu.L of twenty-four phosphate standards (with
phosphate at 2 mM, 1 mM, 500 .mu.M, 250 .mu.M, 125 .mu.M, . . . )
to 10 .mu.L of 100 nM Fl-PBP-Tb (in 10 mM Tris pH 7.6, 0.05% Triton
X-100) in a 384-well plate (Corning 3677). The same series of
reactions was also performed with 100 nM Fl-PBP-Tb that also had
either 10 nM or 100 nM of a Eu-labeled Antibody produced by
standard methods with an amine-reactive TTHA-based Eu-chelate. The
samples were excited at 340 nm (30 nm bandpass) and fluorescence
intensity was captured at 520 nm (25 nm bandpass), 495 nm (10 nm
bandpass), and 615 nm using a 200 .mu.s detection window followed
by a 100 .mu.s delay on a Tecan Ultra instrument. Response as a
function of phosphate concentration was presented either by 520 nm
emission, or the ratio or emission at 520 nm to 615 nm. Results are
presented in FIGS. 13-15.
Sequence CWU 1
1
51346PRTEscherichia coli 1Met Lys Val Met Arg Thr Thr Val Ala Thr
Val Val Ala Ala Thr Leu1 5 10 15Ser Met Ser Ala Phe Ser Val Phe Ala
Glu Ala Ser Leu Thr Gly Ala20 25 30Gly Ala Thr Phe Pro Ala Pro Val
Tyr Ala Lys Trp Ala Asp Thr Tyr35 40 45Gln Lys Glu Thr Gly Asn Lys
Val Asn Tyr Gln Gly Ile Gly Ser Ser50 55 60Gly Gly Val Lys Gln Ile
Ile Ala Asn Thr Val Asp Phe Gly Ala Ser65 70 75 80Asp Ala Pro Leu
Ser Asp Glu Lys Leu Ala Gln Glu Gly Leu Phe Gln85 90 95Phe Pro Thr
Val Ile Gly Gly Val Val Leu Ala Val Asn Ile Pro Gly100 105 110Leu
Lys Ser Gly Glu Leu Val Leu Asp Gly Lys Thr Leu Gly Asp Ile115 120
125Tyr Leu Gly Lys Ile Lys Lys Trp Asp Asp Glu Ala Ile Ala Lys
Leu130 135 140Asn Pro Gly Leu Lys Leu Pro Ser Gln Asn Ile Ala Val
Val Arg Arg145 150 155 160Ala Asp Gly Ser Gly Thr Ser Phe Val Phe
Thr Ser Tyr Leu Ala Lys165 170 175Val Asn Glu Glu Trp Lys Asn Asn
Val Gly Thr Gly Ser Thr Val Lys180 185 190Trp Pro Ile Gly Leu Gly
Gly Lys Gly Asn Asp Gly Ile Ala Ala Phe195 200 205Val Gln Arg Leu
Pro Gly Ala Ile Gly Tyr Val Glu Tyr Ala Tyr Ala210 215 220Lys Gln
Asn Asn Leu Ala Tyr Thr Lys Leu Ile Ser Ala Asp Gly Lys225 230 235
240Pro Val Ser Pro Thr Glu Glu Asn Phe Ala Asn Ala Ala Lys Gly
Ala245 250 255Asp Trp Ser Lys Thr Phe Ala Gln Asp Leu Thr Asn Gln
Lys Gly Glu260 265 270Asp Ala Trp Pro Ile Thr Ser Thr Thr Phe Ile
Leu Ile His Lys Asp275 280 285Gln Lys Lys Pro Glu Gln Gly Thr Glu
Val Leu Lys Phe Phe Asp Trp290 295 300Ala Tyr Lys Thr Gly Ala Lys
Gln Ala Asn Asp Leu Asp Tyr Ala Ser305 310 315 320Leu Pro Asp Ser
Val Val Glu Gln Val Arg Ala Ala Trp Lys Thr Asn325 330 335Ile Lys
Asp Ser Ser Gly Lys Pro Leu Tyr340 3452321PRTEscherichia coli 2Glu
Ala Ser Leu Thr Gly Ala Gly Ala Thr Phe Pro Ala Pro Val Tyr1 5 10
15Ala Lys Trp Ala Asp Thr Tyr Gln Lys Glu Thr Gly Asn Lys Val Asn20
25 30Tyr Gln Gly Ile Gly Ser Ser Gly Gly Val Lys Gln Ile Ile Ala
Asn35 40 45Thr Val Asp Phe Gly Ala Ser Asp Ala Pro Leu Ser Asp Glu
Lys Leu50 55 60Ala Gln Glu Gly Leu Phe Gln Phe Pro Thr Val Ile Gly
Gly Val Val65 70 75 80Leu Ala Val Asn Ile Pro Gly Leu Lys Ser Gly
Glu Leu Val Leu Asp85 90 95Gly Lys Thr Leu Gly Asp Ile Tyr Leu Gly
Lys Ile Lys Lys Trp Asp100 105 110Asp Glu Ala Ile Ala Lys Leu Asn
Pro Gly Leu Lys Leu Pro Ser Gln115 120 125Asn Ile Ala Val Val Arg
Arg Ala Asp Gly Ser Gly Thr Ser Phe Val130 135 140Phe Thr Ser Tyr
Leu Ala Lys Val Asn Glu Glu Trp Lys Asn Asn Val145 150 155 160Gly
Thr Gly Ser Thr Val Lys Trp Pro Ile Gly Leu Gly Gly Lys Gly165 170
175Asn Asp Gly Ile Ala Ala Phe Val Gln Arg Leu Pro Gly Ala Ile
Gly180 185 190Tyr Val Glu Tyr Ala Tyr Ala Lys Gln Asn Asn Leu Ala
Tyr Thr Lys195 200 205Leu Ile Ser Ala Asp Gly Lys Pro Val Ser Pro
Thr Glu Glu Asn Phe210 215 220Ala Asn Ala Ala Lys Gly Ala Asp Trp
Ser Lys Thr Phe Ala Gln Asp225 230 235 240Leu Thr Asn Gln Lys Gly
Glu Asp Ala Trp Pro Ile Thr Ser Thr Thr245 250 255Phe Ile Leu Ile
His Lys Asp Gln Lys Lys Pro Glu Gln Gly Thr Glu260 265 270Val Leu
Lys Phe Phe Asp Trp Ala Tyr Lys Thr Gly Ala Lys Gln Ala275 280
285Asn Asp Leu Asp Tyr Ala Ser Leu Pro Asp Ser Val Val Glu Gln
Val290 295 300Arg Ala Ala Trp Lys Thr Asn Ile Lys Asp Ser Ser Gly
Lys Pro Leu305 310 315 320Tyr310PRTHomo sapiens 3Glu Gln Lys Leu
Ile Ser Glu Glu Asp Leu1 5 1049PRTInfluenza virus 4Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala1 556PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6x His tag 5His His His His His His1
5
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