U.S. patent application number 15/460372 was filed with the patent office on 2017-07-13 for methods of treating diabetes and compositions capable of same.
This patent application is currently assigned to Ramot at Tel-Aviv University Ltd.. The applicant listed for this patent is Ramot at Tel-Aviv University Ltd.. Invention is credited to Yaron BRAM, Ehud GAZIT.
Application Number | 20170198021 15/460372 |
Document ID | / |
Family ID | 44628254 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170198021 |
Kind Code |
A1 |
BRAM; Yaron ; et
al. |
July 13, 2017 |
METHODS OF TREATING DIABETES AND COMPOSITIONS CAPABLE OF SAME
Abstract
A composition of matter is disclosed which comprises isolated
oligomers of human islet amyloid polypeptide (IAPP). Antibodies
recognizing same are also disclosed. Use of the composition of
matter and the antibodies are also disclosed.
Inventors: |
BRAM; Yaron; (Haifa, IL)
; GAZIT; Ehud; (Ramat-HaSharon, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ramot at Tel-Aviv University Ltd. |
Tel-Aviv |
|
IL |
|
|
Assignee: |
Ramot at Tel-Aviv University
Ltd.
Tel-Aviv
IL
|
Family ID: |
44628254 |
Appl. No.: |
15/460372 |
Filed: |
March 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13700444 |
Nov 28, 2012 |
9624285 |
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PCT/IL2011/000436 |
Jun 2, 2011 |
|
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15460372 |
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61350943 |
Jun 3, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/17 20130101;
A61P 5/48 20180101; C07C 305/06 20130101; A61P 3/10 20180101; C07K
16/26 20130101; G01N 2500/10 20130101; G01N 2333/4709 20130101;
A61K 39/0005 20130101; G01N 33/6893 20130101; C07K 14/575
20130101 |
International
Class: |
C07K 14/575 20060101
C07K014/575; C07K 16/26 20060101 C07K016/26; A61K 39/00 20060101
A61K039/00; C07C 305/06 20060101 C07C305/06 |
Claims
1. A composition of matter comprising isolated non-crosslinked
oligomers of human islet amyloid polypeptide (IAPP), wherein the
majority of said oligomers are dimers and/or trimers and wherein
said oligomers are stable for up to 7 days.
2. The composition of matter of claim 1, wherein said oligomers are
stable for up to 7 days.
3. The composition of matter of claim 1, wherein said composition
comprises an anionic surfactant.
4. The composition of matter of claim 3, wherein said anionic
surfactant comprises sodium dodecyl sulfate (SDS).
5. A method of generating a composition of matter comprising
isolated oligomers of human islet amyloid polypeptide (IAPP), the
method comprising: (a) dissolving human IAPP in an agent that
eliminates structured forms of IAPP; (b) removing said agent; and
(c) redissolving said non-structured form of IAPP in a solvent and
an anionic surfactant, thereby generating the composition of
matter.
6. The method of claim 5, wherein said agent is selected from the
group consisting of 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP),
trifluoroethanol (TFE), and trifluoroacetic acid (TFA).
7. The method of claim 5, wherein said solvent is selected from the
group consisting of sodium hydroxide, potassium hydroxide, ammonium
hydroxide, and dimethyl sulfoxide.
8. The method of claim 5, wherein said anionic surfactant is
selected from the group consisting of Sodium dodecyl sulfate (SDS),
Ammonium lauryl sulfate, Docusate sodium salt, N-Lauroylsarcosine
sodium salt, Lithium dodecyl sulfate, 1-Octanesulfonic acid sodium
salt, Sodium 1-butanesulfonate, Sodium hexanesulfonate,
Chenodeoxycholic acid, Dehydrocholic acid, Glycocholic acid and
Sodium deoxycholate.
9. An isolated antibody which binds with a higher affinity to
oligomers of human Islet Amyloid Polypeptide (IAPP) than fibrils of
human IAPP.
10. The isolated antibody of claim 9, attached to an identifiable
moiety.
11. The isolated antibody of claim 9, being a polyclonal
antibody.
12. The isolated antibody of claim 9, being a monoclonal
antibody.
13. The isolated antibody of claim 9 being formulated in a
pharmaceutical composition.
14. A method of detecting IAPP oligomers in a biological sample,
the method comprising contacting the biological sample with the
antibody of claim 9 under conditions which allow formation of
immunocomplexes, wherein a presence of immunocomplexes above a
predetermined threshold is indicative of IAPP oligomers in the
biological sample.
15. A method of diagnosing diabetes in a subject in need thereof,
the method comprising detecting IAPP oligomers in a biological
sample of the subject, wherein a presence or level above a
predetermined threshold of said IAPP oligomers in said biological
sample, is indicative of diabetes in the subject.
16. The method of claim 15, wherein said detecting is effected
using an antibody which binds with a higher affinity to oligomers
of human Islet Amyloid Polypeptide (IAPP) than fibrils of human
IAPP.
17. A method of treating diabetes in a subject in need thereof, the
method comprising administering to the subject a therapeutic
effective amount of an agent which reduces the activity or amount
of an IAPP oligomer, thereby treating diabetes in the subject.
18. The method of claim 17, wherein said agent is an antibody which
binds with a higher affinity to oligomers of human Islet Amyloid
Polypeptide (IAPP) than fibrils of human IAPP.
19. A method of identifying an agent useful for treating diabetes,
the method comprising contacting the agent with the composition of
matter of claim 1, wherein a down-regulation of an amount or
activity of said oligomers is indicative of an agent useful for the
treatment of diabetes.
20. The method of claim 19, wherein the agent is a small molecule
or an antibody.
21. The method of claim 19, wherein said contacting is effected in
the presence of cells.
22. A vaccine comprising the composition of matter of claim 1 and
an immunologically acceptable carrier.
23. A method of treating diabetes in a subject in need thereof, the
method comprising administering to the subject an effective amount
of the vaccine of claim 22, thereby treating diabetes in the
subject.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 13/700,444 filed on Nov. 28, 2012, which is a
National Phase of PCT Patent Application No. PCT/IL2011/000436
having International Filing Date of Jun. 2, 2011, which claims the
benefit of priority under 35 USC .sctn.119(e) of U.S. Provisional
Patent Application No. 61/350,943 filed on Jun. 3, 2010. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 69448SequenceListing.txt, created
on Mar. 16, 2017, comprising 41,879 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to compositions and methods for diagnosing and treating
diabetes.
[0004] The transition of soluble peptides and proteins into
highly-ordered amyloid structures is associated with major human
disorders including Alzheimer's disease (AD), Parkinson's disease,
Prion and Type II Diabetes (T2D). While amyloid fibrils were
previously considered as the main pathological elements that
facilitate the tissue degeneration observed in amyloid-related
disorders, there is an increased body of evidence which suggest a
key role for early soluble oligomeric assemblies in the process of
cytotoxicity and cell death. A comprehensive postmortem study of
AD-associated .beta.-amyloid polypeptide (A.beta.) aggregation
showed poor correlation between amyloid plaque burden and cognitive
functions in elderly population. This lead to the reexamination of
the amyloid hypothesis regarding fibrils as the major toxic species
in amyloid-associated diseases. Since this early work, a number of
studies provided evidence that A.beta. oligomers are in fact
significantly more cytotoxic than mature fibrils. Moreover the
intracranial reintroduction of purified soluble oligomer into the
brain of normal rodents resulted in severe memory impairment.
Revolutionary work has shown that it is possible to generate
soluble toxic oligomeric forms of A.beta. 1-42 in vitro [Lambert,
M. P. et al. Proc. Natl. Acad. Sci. USA. 95, 6448-6453, (1998);
Barghorn, S. et al. J. Neurochem. 95, 834-847, (2005). Braghorn et
al. also demonstrated that although these assemblies were formed in
vitro under relatively harsh conditions similar epitopes were also
observed in vivo in AD patient's brains and in amyloid precursor
protein transgenic mice [J. Neurochem. 95, 834-847, (2005)]. These
epitopes are currently being used for the development of
immunological treatment of AD.
[0005] In addition, several proteins that are not associated with
any known disease can form oligomers-like structures in vitro
[Caughey, B. & Lansbury, P. T. Annu. Rev. Neurosci. 26,
267-298, (2003)]. The observation that these newly formed
structures exhibit toxicity similar to amyloid oligomers, suggest a
much wider mechanism of toxicity not related to amyloid formation.
Amyloid oligomers specifically increase lipid bilayer conductance
regardless of the sequence, whereas fibrils and soluble low
molecular weight species have no observable effect on membranes
[Bucciantini, M. et al. J. Biol. Chem. 279, 31374-31382,
(2004)].
[0006] The islet amyloid polypeptide (IAPP) is a 37 amino acid
peptide hormone, packaged and secreted with insulin by pancreatic
.beta.-cells in secretory granules. Under normal conditions IAPP is
released into the blood circulation and excreted via the renal
system. IAPP is part of the endocrine system and contributes to
glycemic control. This peptide is highly conserved between species,
implying a functional significance. Type 2 Diabetes (T2D) is
characterized by a disruption of insulin secretion from islet
Langerhans cells and decreased insulin sensitivity of peripheral
tissue. The first description of amyloid deposits in pancreatic
islets of a diabetic subject was made more than 100 years ago.
Islet amyloidosis can affect less than 1% or up to 80% of islets in
a diabetic individual. The occurrence of islet amyloid in
non-diabetic subjects is low, less than 15% in elderly, apparently
non-diabetic individuals, but is high in more than 90% of diabetic
subjects postmortem. In 1987, two groups identified the
constitutive protein in islet amyloid, denoting it Amylin or IAPP.
As other amyloid-related diseases, the amyloid deposits in
pancreatic islets were considered as the primary toxic agent and as
a primary cause of pancreatic degeneration for many years.
[0007] In the last decade this dogma was challenged by several
studies suggesting that soluble oligomers may be the primary toxic
species as islet amyloid is also found in non-diabetic individuals,
particularly with elderly population, and is not present in all
islets in people with T2D. Homozygous transgenic mice of human IAPP
(hIAPP) developed severe diabetes due to a high rate of .beta.-cell
apoptosis already at the age of 10 weeks. However, extracellular
islet amyloid was not yet present in these mice during the rapid
loss of .beta.-cells from age 5-10 weeks. In obese hemizygous hIAPP
mice that develop diabetes at approximately 20 weeks of age,
extensive islet amyloid does accrue, but there is a poor
correlation between the extent of islet amyloid and the frequency
of .beta.-cell apoptosis.
[0008] Porat, Y., et al., Biochemistry 42, 10971-10977, (2003)
showed that the soluble structures of the hIAPP peptide interact
and destabilize biological membrane. By showing that inhibition of
IAPP fibrillation by rifampicin did not inhibit toxicity towards
pancreatic cells, Meier et al. [Am. J. Physiol. Endocrinol. Metab.
291, E1317-1324, (2006)] proved that oligomers are probably the
primary toxic epitope in T2D.
[0009] In spite of the extensive clinical importance of amyloid
oligomers formation in T2D, the molecular mechanism that leads to
the self-assembly and molecular recognition process is still not
fully understood and soluble oligomers were never stabilized as
distinctive entities.
[0010] U.S. Patent Application Publication No. 20090246191 teaches
crosslinked prefibrillar aggregates of IAPP and crosslinked forms
of Beta amyloid oligomers.
[0011] Porat et al., [Biochem, 2003, 42, 10971-10977] teaches
pre-fibrillar structures of IAPP having a beta sheet secondary
structure.
SUMMARY OF THE INVENTION
[0012] According to an aspect of some embodiments of the present
invention there is provided a composition of matter comprising
isolated oligomers of human islet amyloid polypeptide (IAPP).
[0013] According to an aspect of some embodiments of the present
invention there is provided a method of generating the composition
of matter of the present invention, the method comprising:
[0014] (a) dissolving human IAPP in an agent that eliminates
structured forms of IAPP;
[0015] (b) removing the agent;
[0016] (c) redissolving the non-structured form of IAPP in a
solvent and an anionic surfactant, thereby generating the
composition of matter of the present invention.
[0017] According to an aspect of some embodiments of the present
invention there is provided a vaccine comprising oligomers of human
IAPP and an immunologically acceptable carrier.
[0018] According to an aspect of some embodiments of the present
invention there is provided a method of treating diabetes in a
subject in need thereof, the method comprising administering to the
subject an effective amount of the vaccine of the present
invention, thereby treating diabetes in the subject.
[0019] According to an aspect of some embodiments of the present
invention there is provided an isolated antibody which binds with a
higher affinity to oligomers of human Islet Amyloid Polypeptide
(IAPP) than fibrils of human IAPP.
[0020] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient the isolated antibody of the present
invention.
[0021] According to an aspect of some embodiments of the present
invention there is provided a method of detecting IAPP oligomers in
a biological sample, the method comprising contacting the
biological sample with the antibody of the present invention under
conditions which allow formation of immunocomplexes, wherein a
presence of immunocomplexes above a predetermined threshold is
indicative of IAPP oligomers in the biological sample.
[0022] According to an aspect of some embodiments of the present
invention there is provided a method of diagnosing diabetes in a
subject in need thereof, the method comprising detecting IAPP
oligomers in a biological sample of the subject, wherein a presence
or level above a predetermined threshold of the IAPP oligomers in
the biological sample, is indicative of diabetes in the
subject.
[0023] According to an aspect of some embodiments of the present
invention there is provided a method of diagnosing diabetes in a
subject in need thereof, the method comprising detecting antibodies
which recognize IAPP oligomers in a biological sample of the
subject, wherein a presence or level above a predetermined
threshold of the antibodies in the biological sample, is indicative
of diabetes in the subject.
[0024] According to an aspect of some embodiments of the present
invention there is provided a method of treating diabetes in a
subject in need thereof, the method comprising administering to the
subject a therapeutic effective amount of an agent which reduces
the activity or amount of an IAPP oligomer, thereby treating
diabetes in the subject.
[0025] According to an aspect of some embodiments of the present
invention there is provided a method of identifying an agent useful
for treating diabetes, the method comprising contacting the agent
with the composition of matter of claim 1, wherein a
down-regulation of an amount or activity of the oligomers is
indicative of an agent useful for the treatment of diabetes.
[0026] According to some embodiments of the invention, the
oligomers comprise dimers and/or trimers.
[0027] According to some embodiments of the invention, the
oligomers have a molecular weight between 4 kDa and 90 kDa.
[0028] According to some embodiments of the invention, the
composition is devoid of fibrils of IAPP.
[0029] According to some embodiments of the invention, the
composition of matter further comprising sodium dodecyl sulfate
(SDS).
[0030] According to some embodiments of the invention, the
oligomers have an alpha helical conformation.
[0031] According to some embodiments of the invention, the
oligomers are crosslinked.
[0032] According to some embodiments of the invention, the
oligomers are non-crosslinked.
[0033] According to some embodiments of the invention, the
oligomers consist of dimers and/or trimers.
[0034] According to some embodiments of the invention, the
composition of matter is stable for up to 7 days.
[0035] According to some embodiments of the invention, the agent is
selected from the group consisting of 1,1,1,3,3,3
hexafluoro-2-propanol (HFIP), trifluoroethanol (TFE), and
trifluoroacetic acid (TFA).
[0036] According to some embodiments of the invention, the solvent
is selected from the group consisting of sodium hydroxide,
potassium hydroxide, ammonium hydroxide, and dimethyl
sulfoxide.
[0037] According to some embodiments of the invention, the anionic
surfactant is selected from the group consisting of Sodium dodecyl
sulfate (SDS), Ammonium lauryl sulfate, Docusate sodium salt,
N-Lauroylsarcosine sodium salt, Lithium dodecyl sulfate,
1-Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium
hexanesulfonate, Chenodeoxycholic acid, Dehydrocholic acid,
Glycocholic acid and Sodium deoxycholate.
[0038] According to some embodiments of the invention, the isolated
antibody is attached to an identifiable moiety.
[0039] According to some embodiments of the invention, the isolated
antibody is a polyclonal antibody.
[0040] According to some embodiments of the invention, the isolated
antibody is a monoclonal antibody.
[0041] According to some embodiments of the invention, the
detecting is effected using the antibody of the present
invention.
[0042] According to some embodiments of the invention, the agent is
the antibody of the present invention.
[0043] According to some embodiments of the invention, the agent is
a small molecule or an antibody.
[0044] According to some embodiments of the invention, the
contacting is effected in the presence of cells.
[0045] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0046] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0047] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings
and images. With specific reference now to the drawings in detail,
it is stressed that the particulars shown are by way of example and
for purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0048] In the drawings:
[0049] FIGS. 1A-1E are graphs and photographs illustrating
characterization of human IAPP oligomers. (FIG. 1A) PAGE analysis
under non-reducing conditions of human IAPP oligomers and a
negative control of non amyloidogenic rat IAPP (I-monomer, II-dimer
and III-trimer). (FIG. 1B) Oligomer stability assay, hIAPP
oligomers were dialyzed against PBS buffer and incubated at
37.degree. C. Oligomer association/disassociation was monitored by
PAGE analysis under non reducing conditions. (FIG. 1C) Size
exclusion chromatography (Superdex 75 10/300, PBS buffer pH 7.4) of
hIAPP oligomers; I-monomer, I-dimer, III-trimer and IV-90 kDa
oligomer. (FIG. 1D) Transmittance electron microscopy (TEM) and
atomic force microscopy (AFM) images of the .about.90 kDa
oligomers, TEM scale bar 100 nm, AFM scale bar 600 nm. (FIG. 1E) CD
spectroscopy of hIAPP and rIAPP, protein concentration of 5 .mu.M.
Each spectrum represents the average of three measurements.
[0050] FIGS. 2A-2D are graphs illustrating the toxicity of human
IAPP oligomers. (FIG. 2A) Rin-m cells treated with hIAPP oligomers
(grey) or with rIAPP (black) in diverse concentrations. Cell
viability was estimated by MTT reduction assay (*P<0.05,
**P<0.005). (FIG. 2B) Dye leakage from calcein containing
liposomes. 1 .mu.M hIAPP oligomers (black squares) or rIAPP (grey
rhombus) were incubated with the liposomes, and membrane damage was
evaluated by increased fluorescence (excitation: 495, emission:
520) and compared to the control group (white rectangle). (FIG. 2C)
FACS results of the incubation of Rin-m cells with hIAPP oligomers
at different concentrations. The Annexin V-FITC apoptosis detection
kit was used for the detection of apoptotic cells. FL1-H is the
fluorescence of V-FITC and FL2-H is the fluorescence of Annexin
V-PE. I--cells in necrotic state, II--late apoptotic state,
III--early apoptotic state and IV--viable cells (FIG. 2D) Diagram
presentation of cell state dispersion of three FACS analysis
assays, dark grey column represent viable cells, light grey column
represent early and late apoptotic cells and black column represent
necrotic cells.
[0051] FIG. 3 are confocal microscopic images illustrating that
hIAPP oligomers permeabilize the cell membrane. Rin-m cells were
incubated with 5 .mu.M of hIAPP-Hiytelfluor 488 oligomers, and
stained with phalloidin-tetramethylrhodamine. Incubation was
performed for one hour (I), four hours (IV) and eight hours (VIII).
After one hour, localization of hIAPP oligomers to the cell
membrane was observed followed by insertion into the cytoplasm.
Cell morphology alterations could be detected following longer
incubation times.
[0052] FIGS. 4A-4E are graphs and photographs illustrating that
antibodies from Type II Diabetes patients recognize and neutralize
hIAPP oligomers. (FIG. 4A) Purified antibodies from serum of type
II diabetes patients and healthy people (N=3) was compared by
analyzing their ability to recognize hIAPP oligomers (5 .mu.g). Dot
blot analysis was performed on serial dilutions of purified
antibodies. (FIG. 4B) Densitometer analysis by Scion image of
recognition properties of purified antibodies to hIAPP oligomers,
light grey columns represent non type II diabetes antibodies and
dark grey columns represents type II diabetes purified antibodies.
(FIG. 4C) Rin-m cells treated with hIAPP oligomers (light grey, 5
.mu.M) alone, Rin-m cells treated with hIAPP oligomers and Type II
diabetes (orange) or non Type II diabetes (dark grey) purified
antibodies in diverse concentration were examined for viability by
MTT reduction assay and compared to the non-treated cells (black),
(*P<0.05, **P<0.005). (FIG. 4D) PAGE analysis and
Western-blot analysis of hIAPP oligomers was performed in order to
study which of the multimers type II diabetes antibodies
recognize., Positive control was performed with rabbit anti IAPP
(Santa Cruz Biotechnology, USA) and negative control was done with
non type II diabetes purified antibodies. (FIG. 4E) Bar graph
illustrating the amount of antibody recognition in Type II diabetic
patients as compared to non-Type II diabetic patients.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0053] The present invention, in some embodiments thereof, relates
to compositions and methods for diagnosing and treating
diabetes.
[0054] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0055] Soluble oligomeric assemblies of amyloid proteins emerge as
the major pathological agent in degenerative misfolding diseases.
Unlike the characterized Alzheimer's disease .beta.-amyloid
oligomers, the oligomeric state of the islet amyloid polypeptide
(IAPP), was never stabilized, probed or manipulated. The present
invention relates to the isolation of stable IAPP cytotoxic
oligomers. These oligomers induce apoptosis in cultured pancreatic
cells (FIGS. 2C-2D), permeate model lipid vesicles (FIG. 3) and
interact with the cell membrane following complete internalization
(FIG. 3). Moreover, antibodies that specifically recognize these
assemblies were exclusively identified in diabetes patients and
were able to neutralize the apoptotic cytotoxic effect of these
oligomers (FIGS. 4A-4E). The present findings shows that human IAPP
oligomers are not only stable and highly toxic to cultured cells,
they are also found in Type II diabetes patients and accordingly
play a major role in the disease progression.
[0056] The present inventors propose that vaccines comprising human
IAPP oligomers may be useful for the treatment of Diabetes as well
as antibodies capable of specifically recognizing the human IAPP
oligomer.
[0057] Thus, according to one aspect of the present invention there
is provided a composition of matter comprising isolated oligomers
of human IAPP.
[0058] Islet amyloid polypeptide (IAPP) is a peptide hormone,
consisting essentially of 37 amino acids, which is synthesized in
the beta cells of the pancreas and which, together with insulin and
glucagon, is involved in the regulation of sugar metabolism. IAPP
is an antagonist of insulin.
[0059] The polynucleotide and polypeptide sequence of IAPP is set
forth in Accession No. NM_000415 (SEQ ID NO: 1), NP_000406 (SEQ ID
NO: 2), NM_010491 (SEQ ID NO: 3) AND NP_034621 (SEQ ID NO: 4).
[0060] As used herein, the term "oligomer" refers to covalent and
non-covalent dimers and/or trimers or higher aggregates of IAPP
that do not form fibrous structures i.e. regular .beta.-sheet array
(e.g. devoid of fibrils of IAPP).
[0061] The phrase "isolated oligomers" refers to the oligomers
being substantially free from other substances (e.g., other
pancreatic cells, blood components, hormones, proteins or nucleic
acids, etc.) that are present in its in-vivo environment.
[0062] The isolated oligomers of the present invention are
typically synthetically produced and are not part of an extract
isolated from the pancreas or blood.
[0063] According to one embodiment, the isolated oligomers of the
present invention comprise an .alpha.-helical secondary structure
with globular morphology.
[0064] According to another embodiment, the molecular weight of the
oligomers of the present invention is between about 4-90 kDa.
[0065] As used herein, the term "fibril" refers to a thread-like
filamentous structure composed of higher ordered aggregates which
is typically visible in an electron microscope.
[0066] Generation of the oligomers of this aspect of the present
invention may be performed by:
[0067] (a) dissolving human IAPP in an agent that eliminates
structured forms of IAPP;
[0068] (b) removing the agent; and
[0069] (c) redissolving the non-structured form of IAPP in a
solvent and an anionic surfactant.
[0070] The peptide human IAPP may be generated by solid phase
peptide synthesis or by recombinant means. Large scale peptide
synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.
Human IAPP is also commercially available--for example from Bachem,
Bubendorf, Switzerland (H-7905).
[0071] Examples of agents which are capable of decreasing the
amount of structure in human IAPP include, but are not limited to
1,1,1,3,3,3 hexafluoro-2-propanol (HFIP), trifluoroethanol (TFE),
and trifluoroacetic acid (TFA).
[0072] Following dissolving of the IAPP, the dissolving agent is
then removed (e.g. by drying, including air drying and drying under
vacuum).
[0073] The non-structured form of IAPP is then redissolved in a
solvent and an ionic surfactant.
[0074] Exemplary solvents that may be used according to this aspect
of the present invention include sodium hydroxide, potassium
hydroxide, ammonium hydroxide, and dimethyl sulfoxide.
[0075] Exemplary anionic surfactants that may be used according to
this aspect of the present invention include Sodium dodecyl sulfate
(SDS), Ammonium lauryl sulfate, Docusate sodium salt,
N-Lauroylsarcosine sodium salt, Lithium dodecyl sulfate,
1-Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium
hexanesulfonate, Chenodeoxycholic acid, Dehydrocholic acid,
Glycocholic acid and Sodium deoxycholate.
[0076] According to a specific embodiment, the agent used to
dissolve IAPP is 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP), the
solvent used to redissolve the non-structured form of IAPP is NaOH
and the anionic surfactant used is SDS.
[0077] The present inventors showed that IAPP oligomers generated
according to the above described protocol are stable for up to 7
days. In order to enhance stability, the oligomers may be
crosslinked. Methods of crosslinking IAPP oligomers are disclosed
in U.S. Patent Application Publication No. 20090246191, the
contents of which are incorporated herein by reference. It will be
appreciated that in the absence of crosslinking, the IAPP oligomers
typically are no greater than trimers, such that compositions
comprising the non-crosslinked oligomers of this aspect of the
present invention are devoid of pentamers and/or hexamers.
[0078] The IAPP oligomers of this aspect of the present invention
may be used for a variety of applications e.g._to isolate and/or
purify oligomer reactive antibodies or fragments thereof from
biological fluids. In another embodiment, the purified IAPP
oligomers may be used to screen for and detect IAPP-oligomer
reactive antibodies or fragments thereof in a biological sample.
The purified oligomers may be used as a ligand in these methods.
The oligomers may also be used as an immunogen to induce production
of antibodies which specifically recognize the oligomeric state of
IAPP.
[0079] Thus, according to another aspect of the present invention
there is provided an isolated antibody which is capable of
specifically binding to IAPP oligomers. Such antibodies bind with a
higher affinity to oligomers of human Islet Amyloid Polypeptide
(IAPP) than fibrils of human IAPP.
[0080] According to one embodiment of this aspect of the present
invention, the antibodies bind with at least a two fold, preferably
at least a 5 fold, even more preferably at least a 10 fold and even
more preferably at least a 20 fold higher affinity for the
oligomeric over the fibrillar form of IAPP.
[0081] According to another embodiment of this aspect of the
present invention, the antibodies bind with the same affinity to
the oligomeric IAPP and to the fibrillar form of IAPP.
[0082] According to still another embodiment of this aspect of the
present invention, the antibodies bind with at least a two fold
higher affinity to IAPP oligomers, more preferably at least a 5
fold higher affinity, more preferably at least a 10 fold higher
affinity, more preferably at least a 20 fold higher affinity than
to other amyloidogenic oligomers (e.g. serum amyloid A protein,
beta2-microglobulin, transthyretin, cystatin C variant, gelsolin,
procalcitonin, PrP protein, amyloid beta-protein, ApoA1, and
lysozyme).
[0083] As used herein, the term "antibody" refers to a
substantially intact antibody molecule or an antibody fragment.
[0084] The phrase "isolated antibody" refers to an antibody which
has been removed from its natural environment. For example, the
present inventors have isolated anti-IAPP oligomer antibodies onto
a filter using a dot-blot assay (see FIG. 4A).
[0085] As used herein, the phrase "antibody fragment" refers to a
functional fragment of an antibody that is capable of binding to an
antigen.
[0086] Suitable antibody fragments for practicing the present
invention include, inter alia, a complementarity-determining region
(CDR) of an immunoglobulin light chain (referred to herein as
"light chain"), a CDR of an immunoglobulin heavy chain (referred to
herein as "heavy chain"), a variable region of a light chain, a
variable region of a heavy chain, a light chain, a heavy chain, an
Fd fragment, and antibody fragments comprising essentially whole
variable regions of both light and heavy chains such as an Fv, a
single-chain Fv, an Fab, an Fab', and an F(ab')2.
[0087] Functional antibody fragments comprising whole or
essentially whole variable regions of both light and heavy chains
are defined as follows:
[0088] (i) Fv, defined as a genetically engineered fragment
consisting of the variable region of the light chain and the
variable region of the heavy chain expressed as two chains;
[0089] (ii) single-chain Fv ("scFv"), a genetically engineered
single-chain molecule including the variable region of the light
chain and the variable region of the heavy chain, linked by a
suitable polypeptide linker;
[0090] (iii) Fab, a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule,
obtained by treating whole antibody with the enzyme papain to yield
the intact light chain and the Fd fragment of the heavy chain,
which consists of the variable and CH1 domains thereof;
[0091] (iv) Fab', a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule,
obtained by treating whole antibody with the enzyme pepsin,
followed by reduction (two Fab' fragments are obtained per antibody
molecule); and
[0092] (v) F(ab')2, a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule,
obtained by treating whole antibody with the enzyme pepsin (i.e., a
dimer of Fab' fragments held together by two disulfide bonds).
[0093] Methods of generating monoclonal and polyclonal antibodies
are well known in the art. Antibodies may be generated via any one
of several known methods, which may employ induction of in vivo
production of antibody molecules, screening of immunoglobulin
libraries (Orlandi, R. et al. (1989). Cloning immunoglobulin
variable domains for expression by the polymerase chain reaction.
Proc Natl Acad Sci USA 86, 3833-3837; and Winter, G. and Milstein,
C. (1991). Man-made antibodies. Nature 349, 293-299), or generation
of monoclonal antibody molecules by continuous cell lines in
culture. These include, but are not limited to, the hybridoma
technique, the human B-cell hybridoma technique, and the
Epstein-Barr virus (EBV)-hybridoma technique (Kohler, G. and
Milstein, C. (1975). Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 256, 495-497; Kozbor, D.
et al. (1985). Specific immunoglobulin production and enhanced
tumorigenicity following ascites growth of human hybridomas. J
Immunol Methods 81, 31-42; Cote R J. et al. (1983). Generation of
human monoclonal antibodies reactive with cellular antigens. Proc
Natl Acad Sci USA 80, 2026-2030; and Cole, S. P. et al. (1984).
Human monoclonal antibodies. Mol Cell Biol 62, 109-120).
[0094] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with the isolated
oligomers of the present invention. Once an immune response is
detected, e.g., antibodies specific for the antigen are detected in
the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well-known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC.TM.. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding the fibrils of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0095] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with the fibrils of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind the fibrils.
[0096] Antibodies also can be generated in vitro, e.g., using
recombinant antibody binding site expressing phage display
libraries, in addition to the traditional in vivo methods using
animals. See, e.g., Huse Science 246:1275 (1989); Ward Nature
341:544 (1989); Hoogenboom Trends Biotechnol. 15:62-70 (1997); Katz
Annu. Rev. Biophys. Biomol. Struct. 26:27-45 (1997).
[0097] In cases where target antigens are too small to elicit an
adequate immunogenic response when generating antibodies in vivo,
such antigens (referred to as "haptens") can be coupled to
antigenically neutral carriers such as keyhole limpet hemocyanin
(KLH) or serum albumin (e.g., bovine serum albumin (BSA)) carriers
(see, for example, U.S. Pat. Nos. 5,189,178 and 5,239,078).
Coupling a hapten to a carrier can be effected using methods well
known in the art. For example, direct coupling to amino groups can
be effected and optionally followed by reduction of the imino
linkage formed. Alternatively, the carrier can be coupled using
condensing agents such as dicyclohexyl carbodiimide or other
carbodiimide dehydrating agents. Linker compounds can also be used
to effect the coupling; both homobifunctional and
heterobifunctional linkers are available from Pierce Chemical
Company, Rockford, Ill., USA. The resulting immunogenic complex can
then be injected into suitable mammalian subjects such as mice,
rabbits, and others. Suitable protocols involve repeated injection
of the immunogen in the presence of adjuvants according to a
schedule designed to boost production of antibodies in the serum.
The titers of the immune serum can readily be measured using
immunoassay procedures which are well known in the art.
[0098] The antisera obtained can be used directly or monoclonal
antibodies may be obtained, as described hereinabove.
[0099] Antibody fragments may be obtained using methods well known
in the art. (See, for example, Harlow, E. and Lane, D. (1988).
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York.) For example, antibody fragments according to the present
invention can be prepared by proteolytic hydrolysis of the antibody
or by expression in E. coli or mammalian cells (e.g., Chinese
hamster ovary (CHO) cell culture or other protein expression
systems) of DNA encoding the fragment.
[0100] Alternatively, antibody fragments can be obtained by pepsin
or papain digestion of whole antibodies by conventional methods. As
described hereinabove, an (Fab')2 antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage
using pepsin produces two monovalent Fab' fragments and an Fc
fragment directly. Ample guidance for practicing such methods is
provided in the literature of the art (for example, refer to: U.S.
Pat. Nos. 4,036,945 and 4,331,647; and Porter, R. R. (1959). The
hydrolysis of rabbit .gamma.-globulin and antibodies with
crystalline papain. Biochem J 73, 119-126). Other methods of
cleaving antibodies, such as separation of heavy chains to form
monovalent light-heavy chain fragments, further cleavage of
fragments, or other enzymatic, chemical, or genetic techniques may
also be used, so long as the fragments retain the ability to bind
to the antigen that is recognized by the intact antibody.
[0101] As described hereinabove, an Fv is composed of paired heavy
chain variable and light chain variable domains. This association
may be noncovalent (see, for example, Inbar, D. et al. (1972).
Localization of antibody-combining sites within the variable
portions of heavy and light chains. Proc Natl Acad Sci USA 69,
2659-2662). Alternatively, as described hereinabove, the variable
domains may be linked to generate a single-chain Fv by an
intermolecular disulfide bond, or alternately such chains may be
cross-linked by chemicals such as glutaraldehyde.
[0102] Preferably, the Fv is a single-chain Fv. Single-chain Fvs
are prepared by constructing a structural gene comprising DNA
sequences encoding the heavy chain variable and light chain
variable domains connected by an oligonucleotide encoding a peptide
linker. The structural gene is inserted into an expression vector,
which is subsequently introduced into a host cell such as E. coli.
The recombinant host cells synthesize a single polypeptide chain
with a linker peptide bridging the two variable domains. Ample
guidance for producing single-chain Fvs is provided in the
literature of the art (see, e.g.: Whitlow, M. and Filpula, D.
(1991). Single-chain Fv proteins and their fusion proteins.
METHODS: A Companion to Methods in Enzymology 2(2), 97-105; Bird,
R. E. et al. (1988). Single-chain antigen-binding proteins. Science
242, 423-426; Pack, P. et al. (1993). Improved bivalent
miniantibodies, with identical avidity as whole antibodies,
produced by high cell density fermentation of Escherichia coli.
Biotechnology (N.Y.) 11(11), 1271-1277; and U.S. Pat. No.
4,946,778).
[0103] Isolated complementarity-determining region peptides can be
obtained by constructing genes encoding the CDR of an antibody of
interest. Such genes may be prepared, for example, by RT-PCR of the
mRNA of an antibody-producing cell. Ample guidance for practicing
such methods is provided in the literature of the art (e.g.,
Larrick, J. W. and Fry, K. E. (1991). PCR Amplification of Antibody
Genes. METHODS: A Companion to Methods in Enzymology 2(2),
106-110).
[0104] It will be appreciated that for human therapy or
diagnostics, humanized antibodies are preferably used. Humanized
forms of non-human (e.g., murine) antibodies are genetically
engineered chimeric antibodies or antibody fragments having
(preferably minimal) portions derived from non-human antibodies.
Humanized antibodies include antibodies in which the CDRs of a
human antibody (recipient antibody) are replaced by residues from a
CDR of a non-human species (donor antibody), such as mouse, rat, or
rabbit, having the desired functionality. In some instances, the Fv
framework residues of the human antibody are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues found neither in the recipient antibody nor in
the imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDRs correspond to those of a non-human antibody and all or
substantially all of the framework regions correspond to those of a
relevant human consensus sequence. Humanized antibodies optimally
also include at least a portion of an antibody constant region,
such as an Fc region, typically derived from a human antibody (see,
for example: Jones, P. T. et al. (1986). Replacing the
complementarity-determining regions in a human antibody with those
from a mouse. Nature 321, 522-525; Riechmann, L. et al. (1988).
Reshaping human antibodies for therapy. Nature 332, 323-327;
Presta, L. G. (1992b). Curr Opin Struct Biol 2, 593-596; and
Presta, L. G. (1992a). Antibody engineering. Curr Opin Biotechnol
3(4), 394-398).
[0105] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
imported residues, which are typically taken from an imported
variable domain. Humanization can be performed essentially as
described (see, for example: Jones et al. (1986); Riechmann et al.
(1988); Verhoeyen, M. et al. (1988). Reshaping human antibodies:
grafting an antilysozyme activity. Science 239, 1534-1536; and U.S.
Pat. No. 4,816,567), by substituting human CDRs with corresponding
rodent CDRs. Accordingly, humanized antibodies are chimeric
antibodies, wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies may be
typically human antibodies in which some CDR residues and possibly
some framework residues are substituted by residues from analogous
sites in rodent antibodies.
[0106] Human antibodies can also be produced using various
additional techniques known in the art, including phage-display
libraries (Hoogenboom, H. R. and Winter, G. (1991). By-passing
immunisation. Human antibodies from synthetic repertoires of
germline VH gene segments rearranged in vitro. J Mol Biol 227,
381-388; Marks, J. D. et al. (1991). By-passing immunization. Human
antibodies from V-gene libraries displayed on phage. J Mol Biol
222, 581-597; Cole et al. (1985), Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96; and Boerner, P. et al.
(1991). Production of antigen-specific human monoclonal antibodies
from in vitro-primed human splenocytes. J Immunol 147, 86-95).
Humanized antibodies can also be created by introducing sequences
encoding human immunoglobulin loci into transgenic animals, e.g.,
into mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon antigenic challenge,
human antibody production is observed in such animals which closely
resembles that seen in humans in all respects, including gene
rearrangement, chain assembly, and antibody repertoire. Ample
guidance for practicing such an approach is provided in the
literature of the art (for example, refer to: U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks, J. D. et al. (1992). By-passing immunization:
building high affinity human antibodies by chain shuffling.
Biotechnology (N.Y.) 10(7), 779-783; Lonberg et al., 1994. Nature
368:856-859; Morrison, S. L. (1994). News and View: Success in
Specification. Nature 368, 812-813; Fishwild, D. M. et al. (1996).
High-avidity human IgG kappa monoclonal antibodies from a novel
strain of minilocus transgenic mice. Nat Biotechnol 14, 845-851;
Neuberger, M. (1996). Generating high-avidity human Mabs in mice.
Nat Biotechnol 14, 826; and Lonberg, N. and Huszar, D. (1995).
Human antibodies from transgenic mice. Int Rev Immunol 13,
65-93).
[0107] After antibodies have been obtained, they may be tested for
activity, for example via enzyme-linked immunosorbent assay
(ELISA).
[0108] The antibodies of this aspect of the present invention may
be attached to a functional moiety, such as a detectable moiety.
Such antibodies may be useful for identifying IAPP oligomers.
[0109] Various types of detectable or reporter moieties may be
conjugated to the antibody of the invention. These include, but not
are limited to, a radioactive isotope (such as .sup.[125]iodine), a
phosphorescent chemical, a chemiluminescent chemical, a fluorescent
chemical (fluorophore), an enzyme, a fluorescent polypeptide, an
affinity tag, and molecules (contrast agents) detectable by
Positron Emission Tomography (PET) or Magnetic Resonance Imaging
(MRI).
[0110] Examples of suitable fluorophores include, but are not
limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC),
Cy-chrome, rhodamine, green fluorescent protein (GFP), blue
fluorescent protein (BFP), Texas red, PE-Cy5, and the like. For
additional guidance regarding fluorophore selection, methods of
linking fluorophores to various types of molecules see Richard P.
Haugland, "Molecular Probes: Handbook of Fluorescent Probes and
Research Chemicals 1992-1994", 5th ed., Molecular Probes, Inc.
(1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.; Hermanson,
"Bioconjugate Techniques", Academic Press New York, N.Y. (1995);
Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al., 1996.
Biochemistry 35:937; Gakamsky D. et al., "Evaluating Receptor
Stoichiometry by Fluorescence Resonance Energy Transfer," in
"Receptors: A Practical Approach," 2nd ed., Stanford C. and Horton
R. (eds.), Oxford University Press, U K. (2001); U.S. Pat. No.
6,350,466 to Targesome, Inc.]. Fluorescence detection methods which
can be used to detect the antibody when conjugated to a fluorescent
detectable moiety include, for example, fluorescence activated flow
cytometry (FACS), immunofluorescence confocal microscopy,
fluorescence in-situ hybridization (FISH) and fluorescence
resonance energy transfer (FRET).
[0111] Numerous types of enzymes may be attached to the antibody of
the invention [e.g., horseradish peroxidase (HPR),
beta-galactosidase, and alkaline phosphatase (AP)] and detection of
enzyme-conjugated antibodies can be performed using ELISA (e.g., in
solution), enzyme-linked immunohistochemical assay (e.g., in a
fixed tissue), enzyme-linked chemiluminescence assay (e.g., in an
electrophoretically separated protein mixture) or other methods
known in the art [see e.g., Khatkhatay M I. and Desai M., 1999. J
Immunoassay 20:151-83; Wisdom G B., 1994. Methods Mol Biol.
32:433-40; Ishikawa E. et al., 1983. J Immunoassay 4:209-327;
Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs A
H. and van Weemen B K., 1980. J Immunoassay 1:229-49).
[0112] The affinity tag (or a member of a binding pair) can be an
antigen identifiable by a corresponding antibody [e.g., digoxigenin
(DIG) which is identified by an anti-DIG antibody) or a molecule
having a high affinity towards the tag [e.g., streptavidin and
biotin]. The antibody or the molecule which binds the affinity tag
can be fluorescently labeled or conjugated to enzyme as described
above.
[0113] Various methods, widely practiced in the art, may be
employed to attach a streptavidin or biotin molecule to the
antibody of the invention. For example, a biotin molecule may be
attached to the antibody of the invention via the recognition
sequence of a biotin protein ligase (e.g., BirA) as described in
the Examples section which follows and in Denkberg, G. et al.,
2000. Eur. J. Immunol. 30:3522-3532. Alternatively, a streptavidin
molecule may be attached to an antibody fragment, such as a single
chain Fv, essentially as described in Cloutier S M. et al., 2000.
Molecular Immunology 37:1067-1077; Dubel S. et al., 1995. J Immunol
Methods 178:201; Huston J S. et al., 1991. Methods in Enzymology
203:46; Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas
6:93; Kipriyanov S M. et al., 1996. Protein Engineering 9:203;
Pearce L A. et al., 1997. Biochem Molec Biol Intl
42:1179-1188).
[0114] Functional moieties, such as fluorophores, conjugated to
streptavidin are commercially available from essentially all major
suppliers of immunofluorescence flow cytometry reagents (for
example, Pharmingen or Becton-Dickinson).
[0115] According to some embodiments of the invention, biotin
conjugated antibodies are bound to a streptavidin molecule to form
a multivalent composition (e.g., a dimer or tetramer form of the
antibody).
[0116] Table 1 provides non-limiting examples of identifiable
moieties which can be conjugated to the antibody of the
invention.
TABLE-US-00001 TABLE 1 Amino Acid Nucleic sequence Acid sequence
Identifiable (GenBank (GenBank Moiety Accession No.) Accession No.)
Green AAL33912 AF435427 Fluorescent (SEQ ID NO: 5) (SEQ ID NO: 6)
protein Alkaline AAK73766 AY042185 phosphatase (SEQ ID NO: 7) (SEQ
ID NO: 8) Peroxidase CAA00083 A00740 (SEQ ID NO: 9) (SEQ ID NO: 10)
Histidine Amino acids Nucleotides tag 264-269 of 790-807 of GenBank
GenBank Accession No. Accession No. AAK09208 AF329457 (SEQ ID NO:
11) (SEQ ID NO: 12) Myc tag Amino acids Nucleotides 273-283 of
817-849 of GenBank GenBank Accession No. Accession No. AAK09208
AF329457 (SEQ ID NO: 11) (SEQ ID NO: 12) Biotin LHHILDAQKMVWNHR/
lygase tag (SEQ ID NO: 13) orange AAL33917 AF435432 fluorescent
(SEQ ID NO: 14) (SEQ ID NO: 15) protein Beta ACH42114 EU626139
galactosidase (SEQ ID NO: 16) (SEQ ID NO: 17) Streptavidin AAM49066
AF283893 (SEQ ID NO: 18) (SEQ ID NO: 19)
[0117] As mentioned, the antibodies of the present invention may be
used for detection of oligomeric IAPP in a biological sample.
[0118] Thus, according to another aspect of the present invention
there is provided a method of detecting IAPP oligomers in a
biological sample, the method comprising contacting the biological
sample with the antibody of the present invention under conditions
which allow formation of immunocomplexes, wherein a presence of
immunocomplexes above a predetermined threshold is indicative of
IAPP oligomers in the biological sample.
[0119] Biological sample may include tissues (e.g. pancreatic
islets), cells, extracellular matrix, and biological fluids.
Biological fluids include but are not limited to blood, plasma,
serum, cerebrospinal fluid, urine, peritoneal fluid, and
saliva.
[0120] According to this aspect the contacting may be effected in
vitro, ex vivo or in vivo.
[0121] The immunocomplex of the present invention can be formed at
a variety of temperatures, salt concentration and pH values and
those of skills in the art are capable of adjusting the conditions
suitable for the formation of each immunocomplex.
[0122] Determining a presence or level of the immunocomplex of the
invention is dependent on the detectable moiety to which the
antibody is attached, essentially as described hereinabove.
[0123] Since the present inventors have shown that oligomeric IAPP
is associated with Diabetes, it follows that the antibodies may
therefore be used for the diagnosis of Diabetes.
[0124] Thus, according to another aspect of the present invention
there is provided a method of diagnosing Diabetes in a subject in
need thereof, the method comprising detecting IAPP oligomers in a
biological sample of the subject, wherein a presence or level above
a predetermined threshold of the IAPP oligomers in the biological
sample is indicative of Diabetes in the subject.
[0125] As mentioned herein above, the present inventors have shown
that Diabetic patients generate antibodies which recognize
oligomeric IAPP, whereas such antibodies are absent in non-diabetic
patients. The present inventors thus propose that such antibodies
may be used as a marker for Diabetes.
[0126] Thus, according to still another aspect of the present
invention there is provided a method of diagnosing diabetes in a
subject in need thereof, the method comprising detecting antibodies
which recognize IAPP oligomers in a biological sample of the
subject, wherein a presence or level above a predetermined
threshold of the antibodies in the biological sample, is indicative
of diabetes in the subject.
[0127] As used herein the term "diagnosing" refers to confirming
the presence of Diabetes, classifying Diabetes, determining a
severity of Diabetes (grade or stage), monitoring Diabetes
progression, forecasting an outcome of Diabetes and/or prospects of
recovery.
[0128] The subject may be a healthy subject (e.g., human)
undergoing a routine well-being check up. Alternatively, the
subject may be at risk of having Diabetes (e.g., a genetically
predisposed subject, a subject with medical and/or family history
of Diabetes) and/or a subject who exhibits suspicious clinical
signs of Diabetes [e.g., a presence of sugar in the urine).
[0129] According to one embodiment, the level of immunocomplex
(IAPP oligomer-antibody) is compared to a control sample from a
non-diseased subject, wherein an up-regulation of immunocomplex
formation is indicative of Diabetes. Preferably, the subject is of
the same species e.g. human, preferably matched with the same age,
weight, sex etc. It will be appreciated that the control sample may
also be of the same subject from a healthy tissue, prior to disease
progression or following disease remission.
[0130] The contacting may be ex vivo (from a sample removed from
the subject) or in vivo.
[0131] Since the present inventors have now ascertained that it is
the oligomeric form of IAPP that is toxic to islets, the present
inventors propose that an agent which is capable of reducing the
activity or amount of IAPP may be used for the treatment of
Diabetes.
[0132] Thus, according to yet another aspect of the present
invention there is provided a method of treating Diabetes in a
subject in need thereof, the method comprising administering to the
subject a therapeutic effective amount of an agent which reduces
the activity or amount of an IAPP oligomer, thereby treating
diabetes in the subject.
[0133] As used herein "Diabetes" refers to a disease resulting
either from an absolute deficiency of insulin (type 1 diabetes) due
to a defect in the biosynthesis or production of insulin, or a
relative deficiency of insulin in the presence of insulin
resistance (type 2 diabetes), i.e., impaired insulin action, in an
organism. The diabetic patient thus has absolute or relative
insulin deficiency, and displays, among other symptoms and signs,
elevated blood glucose concentration, presence of glucose in the
urine and excessive discharge of urine.
[0134] According to a particular embodiment, the method is for
treating type II Diabetes.
[0135] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0136] According to one embodiment, the agents reduce the amount or
activity of IAPP oligomers by at least 1.25 fold, more preferably
by at least 1.5 fold, more preferably by at least 2 fold and even
more preferably by at least 5 fold.
[0137] Agents which reduce the amount or activity of IAPP oligomers
include but are not limited to antibodies directed against the
oligomers, polynucleotide agents, peptide agents and small molecule
agents.
[0138] Selection of agents which down-regulate the amount of IAPP
oligomers may be performed by contacting the candidate agent and
the isolated IAPP oligomers of the present invention, wherein a
down-regulation of an amount of the oligomers is indicative of an
agent useful for the treatment of diabetes.
[0139] The amount of IAPP oligomers may be estimated using any
protein quantification method which are known in the art.
Visualization of the oligomers may be effected by Page analysis, by
size exclusion chromatography, circular dichroism spectroscopy,
transmission electronic microscopy and atomic force microscopy.
[0140] In addition, the amount of IAPP oligomers may be estimated
by analyzing their activity, as described herein below.
[0141] Selection of agents which down-regulate the activity of IAPP
oligomers may be performed by contacting the candidate agent and
the isolated IAPP oligomers of the present invention with cells (or
artificial cells as described herein below), wherein a
down-regulation of an activity of the oligomers is indicative of an
agent useful for the treatment of diabetes.
[0142] The phrase "reduction of activity of IAPP oligomers" refers
to reduction of the cytotoxic activity of the oligomers.
[0143] Contacting cells with the candidate agent and oligomers can
be performed by any in vitro conditions including for example,
adding the candidate agent and oligomers to cells derived from a
subject (e.g., a primary cell culture, a cell line) or to a
biological sample comprising same (e.g., a fluid, liquid which
comprises the cells) such that both the candidate agent and the
oligomers are in direct contact with the cells. According to some
embodiments of the invention, the cells of the subject are
incubated with the agent and oligomers. The conditions used for
incubating the cells are selected for a time period/concentration
of cells/concentration of drug/ratio between cells and candidate
agent/oligomers and the like which enable the candidate agent to
induce changes to the oligomers.
[0144] Methods of monitoring cellular changes induced by the drugs
are known in the art and include for example, the MTT test which is
based on the selective ability of living cells to reduce the yellow
salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide) (Sigma, Aldrich St Louis, Mo., USA) to a purple-blue
insoluble formazan precipitate; the BrDu assay [Cell Proliferation
ELISA BrdU colorimetric kit (Roche, Mannheim, Germany]; the TUNEL
assay [Roche, Mannheim, Germany]; the Annexin V assay
[ApoAlert.RTM. Annexin V Apoptosis Kit (Clontech Laboratories,
Inc., CA, USA)]; the Senescence associated-.beta.-galactosidase
assay (Dimri G P, Lee X, et al. 1995. A biomarker that identifies
senescent human cells in culture and in aging skin in vivo. Proc
Natl Acad Sci USA 92:9363-9367); as well as various RNA and protein
detection methods (which detect level of expression and/or
activity) which are further described hereinabove.
[0145] As described in the Examples section herein below, the
cytotoxic activity of the IAPP oligomers may also be analyzed using
synthetic membranes--e.g. liposomes, since the ability to penetrate
a cell membrane is thought to be directly correlated with IAPP
cytotoxic activity.
[0146] Agents which are capable of down-regulating the amount or
activity of the IAPP oligomers may be provided to the subject per
se, or as part of a pharmaceutical composition where it is mixed
with a pharmaceutically acceptable carrier.
[0147] As used herein a `pharmaceutical composition` refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to the
subject treated.
[0148] Herein the term `active ingredient` refers to the compound,
which is accountable for the biological effect.
[0149] Hereinafter, the phrases `physiologically acceptable
carrier` and `pharmaceutically acceptable carrier` which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to the subject and does not abrogate
the biological activity and properties of the administered
compound. Preferred carriers of the pharmaceutical composition of
the present invention include, but are not limited to, polyethylene
glycol (PEG), a biocompatible polymer with a wide range of
solubility in both organic and aqueous media (Mutter et al.
(1979).
[0150] Herein the term `excipient` refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0151] Techniques for formulation and administration of drugs may
be found in `Remington's Pharmaceutical Sciences,` Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0152] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0153] Alternately, one may administer a preparation in a local
rather than systemic manner, for example, via injection of the
preparation directly into a specific region of a patient's body
(e.g. pancreas or liver).
[0154] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0155] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0156] For injection, the active ingredients of the invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0157] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries if desired,
to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable
polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0158] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0159] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0160] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0161] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0162] The preparations described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0163] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0164] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0165] The preparation of the present invention may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0166] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients effective to prevent,
alleviate or ameliorate symptoms of disease or prolong the survival
of the subject being treated.
[0167] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art.
[0168] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro assays. For example, a dose can be
formulated in animal models and such information can be used to
more accurately determine useful doses in humans.
[0169] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. [See
e.g., Fingl, et al., (1975) "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1].
[0170] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0171] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0172] Compositions including the preparation of the present
invention formulated in a compatible pharmaceutical carrier may
also be prepared, placed in an appropriate container, and labeled
for treatment of an indicated condition.
[0173] According to another aspect of the present invention, there
is provided an article-of-manufacture including a packaging
material and a pharmaceutical composition identified for treating
amyloid associated diseases (e.g. Diabetes) being contained within
the packaging material, the pharmaceutical composition including,
as an active ingredient, the compound described hereinabove, and a
pharmaceutically acceptable carrier.
[0174] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0175] It will be appreciated that the use of other agents (e.g.,
insulin) and diets (low sugar) can be used in combination with the
agents of the present invention to increase therapeutic efficacy
thereof.
[0176] Since the present inventors found antibodies to the IAPP
oligomers in the serum of Diabetic patients, the present inventors
conceive that the IAPP oligomers may be used as a vaccine for the
treatment of Diabetes, so as to induce generation of antibodies in
vivo.
[0177] Thus, according to another aspect of the present invention
there is provided a vaccine comprising oligomers of human IAPP and
an immunologically acceptable carrier.
[0178] General methods to prepare immunogenic or vaccine
compositions are described in Remington's Pharmaceutical Science;
Mack Publishing Company Easton, Pa. (latest edition). To increase
immunogenicity, the polypeptides of the present invention may be
adsorbed to or conjugated to beads such as latex or gold beads,
ISCOMs, and the like. Immunogenic compositions may comprise
adjuvants, which are substance that can be added to an immunogen or
to a vaccine formulation to enhance the immune-stimulating
properties of the immunogenic moiety. Liposomes are also considered
to be adjuvants (Gregoriades, G. et al., Immunological Adjuvants
and Vaccines, Plenum Press, New York, 1989) Examples of adjuvants
or agents that may add to the effectiveness of proteinaceous
immunogens include aluminum hydroxide, aluminum phosphate, aluminum
potassium sulfate (alum), beryllium sulfate, silica, kaolin,
carbon, water-in-oil emulsions, and oil-in-water emulsions. One
type of adjuvant is muramyl dipeptide (MDP) and various MDP
derivatives and formulations, e.g.,
N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isog-
lutami-ne (GMDP) (Hornung, R L et al. Ther Immunol 1995 2:7-14) or
ISAF-1 (5% squalene, 2.5% pluronic L121, 0.2% Tween 80 in
phosphate-buffered solution with 0.4 mg of threonyl-muramyl
dipeptide; see Kwak, L W et al. (1992) N. Engl. J. Med.,
327:1209-1238). Other useful adjuvants are, or are based on,
cholera toxin, bacterial endotoxin, lipid X, whole organisms or
subcellular fractions of the bacteria Propionobacterium acnes or
Bordetella pertussis, polyribonucleotides, sodium alginate,
lanolin, lysolecithin, vitamin A, saponin and saponin derivatives
such as QS21 (White, A. C. et al. (1991) Adv. Exp. Med. Biol.,
303:207-210) which is now in use in the clinic (Helling, F et al.
(1995) Cancer Res., 55:2783-2788; Davis, T A et al. (1997) Blood,
90: 509), levamisole, DEAE-dextran, blocked copolymers or other
synthetic adjuvants. A number of adjuvants are available
commercially from various sources, for example, Merck Adjuvant 65
(Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
Mich.), Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide),
or a mixture of Amphigen and Alhydrogel. Aluminum is approved for
human use.
[0179] The present invention also contemplates therapeutic
compositions and methods comprising antibodies or an antiserum
induced in one subject using the peptides of the present invention,
removed from that subject and used to treat another subject by
passive immunization or transfer of the antibodies.
[0180] The amount of IAPP oligomer to be administered depends on
the health and weight of the recipient, the route of
administration, the existence of other concurrent treatment, if
any, the frequency of treatment, the nature of the effect desired,
and the judgment of the skilled practitioner.
[0181] An exemplary dose for treating a subject is an amount of up
to about 100 milligrams of oligomer per kilogram of body weight. A
typical single dosage of the oligomer is between about 1 ng and
about 100 mg/kg body weight, and preferably from about 10 .mu.g to
about 50 mg/kg body weight. A total daily dosage in the range of
about 0.1 milligrams to about 7 grams is preferred for intravenous
administration. A useful dose of an antibody for passive
immunization is between 10-100 mg/kg. It has been suggested that an
effective in vivo dose of an antibody/antiserum is between about
10- and 100-fold more than an effective neutralizing concentration
or dose in vitro. These dosages can be determined empirically in
conjunction with the present disclosure and state-of-the-art. The
oligomers of the present invention may be administered alone or in
conjunction with other therapeutics directed to the treatment of
the disease or condition.
[0182] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0183] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0184] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0185] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0186] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Materials and Methods
[0187] Oligomer Preparation:
[0188] IAPP synthetic peptide (Human; H-7905, Bachem, Bubendorf,
Switzerland. Rat; 74-5-10A, American peptide, California, U.S.A)
was suspended in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) at 1
mg/ml and incubated for complete solubilization under shaking (100
rpm) at 37.degree. C. for 2 hours. HFIP was removed by Speedvac
apparatus and peptide was resuspended in 0.2 .mu.M NaOH to a final
concentration of 5 mM, sonicated for 2 minutes in a pre-chilled
sonication bath to ensure complete solubilization. Peptide was
diluted in phosphate-buffered saline (PBS) with 1% Sodium Dodecyl
Sulfate (SDS) to a final concentration of 600 .mu.M and incubated
for 4-7 hours, at 37.degree. C. Peptide was further diluted in
ultra pure H.sub.2O to a final concentration of 200 .mu.M and
incubated for 12 hours, 37.degree. C. IAPP self-assembly products
were analyzed by 15% Tris-tricine PAGE and stained with Imperial
protein stain. For cell and liposome experiments, oligomers were
precipitated by a nine fold excess (v/v) of ice-cold
methanol/acetic acid solution (33% methanol, 4% acetic acid) for 1
hour at 4.degree. C. The oligomers was then pelleted (10 min at
16,200 g), resuspended in PBS buffer, pH 7.4. In order to make sure
SDS is removed entirely, samples were dialyzed against PBS buffer
over night. IAPP oligomers were examined after treatment by PAGE
analysis and size exclusion chromatography. They showed no change
in size distribution.
[0189] Size Exclusion Chromatography:
[0190] IAPP oligomers (0.1 mg) were loaded on Superdex 75 column
10/300 (Amersham Biosciences, Sweden), 0.5 ml/min, phosphate-buffer
saline (PBS). Size was determined using a calibration curve
calculated with 5 protein standard (Bio-Rad. USA). Peaks
Deconvolution was calculated by PeakFit software (SYSTAT software
Inc.).
[0191] Transmission Electronic Microscopy:
[0192] TEM experiments were performed using a JEOL JEM1200EX
microscope operating at 120 kV. Oligomer solution (8 .mu.l) was
placed on a 300 mesh formvar-coated grid and after 2 minutes, the
excess fluid was removed with a filter paper. The samples were
negatively stained with 8 .mu.l uranyl acetate 1% for 2 min.
[0193] Atomic Force Microscopy:
[0194] AFM analysis was generated by depositing an aliquot of 40
.mu.l on a freshly cleaved mica surface. Samples were probed by a
Digital Instrument (DI) MultiMode.TM. NanoScope IV AFM, using a
Mikromasch NSC15/Si3N4 cantilever (resonant frequency f=325 kHz,
spring constant k=40 N/m) in a tapping mode.
[0195] Circular Dichroism (CD) Spectroscopy:
[0196] CD spectra were obtained using an AVIV 202
spectropolarimeter equipped with a temperature-controlled sample
holder and a 10 mm path length cuvette.
[0197] All experiments were performed in PBS, pH 7.4, peptides
concentration of 5 .mu.M. For wavelength scan experiments, each
spectrum represents the average of three scans. Evaluation of the
secondary structure composition obtained from far-UV CD spectra was
facilitated by using the K2d and CDNN software.
[0198] Liposome Membrane Damage Measurements:
[0199] Phosphatidyl ethanolamine, Phosphatidyl serine and
phosphatidyl choline (Avanti, USA) in a molar ratio of 5:3:2
respectively, were dissolved in chloroform at a concentration of 20
mg/ml. Solvent was removed from the sample by evaporating the
chloroform under a stream of nitrogen gas in a rotor vapor
apparatus to deposit a thin lipid film on the walls of a glass test
tube. The dry lipid film was then rehydrated in the 50 mM sodium
phosphate buffer (pH 7.4) containing 40 mM sodium calcein to make
multilamellar vesicles (MLVs) at a concentration of 40 mg/ml. The
MLVs were then subjected to several sonication cycles to
equilibrate the vesicles with the buffer. Nonencapsulated calcein
was removed from vesicles through size exclusion chromatography
using a HiPrep 16/60 sephacryl S-100 column (Amersham Pharmacia
Biotech, Uppsala, Sweden). packed vesicles with calcein were
confirmed by fluorescent measurements before and after adding 1% of
triton x-100. Samples were incubated at 37.degree. C. and the
membrane damage rate was followed by Fluorescence assay (excitation
at 495 nm, 2.5 nm slit, and emission at 520 nm, 5 nm slit).
Measurements were taken using a Jobin Yvon Horiba Fluoromax-3
fluorimeter. Each point represents the average of three independent
measurements.
[0200] MTT Reduction Assay:
[0201] Rin-m cells (2.times.10.sup.5 cells/ml) were cultured in
96-well micro plates (100 .mu.l/well) and incubated overnight at
37.degree. C. Human oligomers and rat IAPP was added to each well
at various concentrations. Each measurement was repeated four
times. Following incubation for 6 hours at 37.degree. C., cell
viability was evaluated using
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)
assay. Briefly, 20 .mu.L of 5 mg/ml MTT dissolved in PBS was added
to each well. After 4 hours incubation at 37.degree. C., 100 .mu.l
of extraction buffer [20% SDS dissolved in a solution of 50%
dimethylformamide and 50% DDW (pH 4.7)] was added to each well, and
the plates were incubated again overnight at 37.degree. C. Finally,
color intensity was measured using an ELISA plate reader at 570
nm.
[0202] Flow Cytometry Cell Sorting (FACS) Assay:
[0203] Rin-m cells (5.times.10.sup.5 cells/ml) were cultured for 4
hours at 37.degree. C. incubated with human IAPP oligomers (final
concentration of 0.5 .mu.M, 1 .mu.M and 5 .mu.M). Samples were
washed with PBS buffer and resuspended with 500 .mu.l binding
buffer. 5 .mu.l of Annexin V-FITC and 10 .mu.l of propidium iodide
(Annexin V-FITC apoptosis detection kit; MBL) was added to the
samples. After 10 minutes of incubation in the dark at RT, samples
were analyzed using the FACS Sort (Beckton Dickinson) and results
analyzed using the CellQuest program (Beckton Dickinson). Each
measurement was repeated three times. FL1-H represents the
fluorescence of V-FITC and FL2-H represents the fluorescence of
annexin V-PE.
[0204] Confocal Microscopy:
[0205] Rin-m cells were cultured on glass cover slips located in
24-well micro plates, then incubated for different periods with
hIAPP-Hiytelfluor 488 (Anaspec, USA) oligomers (5 .mu.M) at
37.degree. C., as described in the cell cytotoxicity experiments
section. After incubation cells were washed with PBS buffer and
fixed with 4% paraformaldehyde in PBS for 5 minutes and washed with
PBS buffer. Cells were treated with 1% Triton x-100 in PBS and
stained with 50 .mu.g/ml Phalloidin Tetramethyl-rhodamine B
isothiocyanate (Sigma-Aldrich) in PBS 40 minutes in room
temperature, followed by extensive wash with PBS buffer. The cells
were imaged using LSM 510 confocal laser scanning microscope (Carl
Zeiss Jena, Germany).
[0206] Antibody Purification:
[0207] Antibodies from three healthy and three type II diabetes
patients serum (Bioreclamation, USA) were purified by protein A
column (GE healthcare). 1 ml of Human Serum was diluted 1:20 with
loading buffer (20 mM Na.sub.2HPO.sub.4, 2 mM NaH.sub.2PO.sub.4
pH=7) and loaded onto a 5-ml protein A column flow throw was
collected and reloaded 3 times. Bound antibody was eluted with 0.1
M of citric acid (pH 3.0) and neutralized with 1 M Tris-HCl (pH
9.0) for 1 ml of eluate 200 .mu.l of Tris buffer was added.
Protein-containing fractions were combined, dialyzed against 2
liter PBS buffer (16 h, 4.degree. C.), antibodies concentration was
determined using Bradford reagent (Sigma-Aldrich).
[0208] Antibody Recognition Assay:
[0209] In order to examine antibody recognition, hIAPP oligomers (5
.mu.g) were applied via a vacuum manifold onto a nitrocellulose
membrane. After blocking the membrane with 5% milk in TBS-T (50 mM
tris, 150 mM NaCl pH=7.5 with 0.3% tween 80) for 1 hour at room
temperature, the membrane was washed briefly with TBS and incubated
with purified antibodies at several concentrations for 2 hours,
room temperature. Then, the membrane was washed briefly with TBS-T
and incubated with HRP-conjugated donkey anti human HRP antibody
(Jackson immunoResearch laboratories, USA). The membrane was
developed using ECL reagents (NEN, USA) according to the supplier's
instructions or 3,3',5,5'-tetramethylbenzidine (TMB). Positive
control was done with rabbit anti IAPP antibody (Santa Cruz
Biotechnology, USA). Bound antibodies were quantified by Scion
image densitometry software (Scion Corporation, USA). In order to
examine which of the assemblies' type II diabetes antibodies
recognize, hIAPP oligomers were separated by 15% Tris-tricine PAGE
and blotted on a nitrocellulose membrane by Trans-Blot semi-dry
transfer cell (Bio-Rad, USA). Binding assay was done the same as
the dot-blot assay with 5 .mu.m/ml antibodies concentration.
[0210] Antibody Neutralizing Effect:
[0211] Rin-m cells (2.times.10.sup.5 cells/ml) were cultured in
96-well micro plates (100 .mu.l/well) and incubated overnight at
37.degree. C. Human oligomers (5 .mu.M) with or without antibodies
was added to each well at various concentrations. Each measurement
was repeated four times; also, a control measurement with
antibodies alone at the highest concentration was preformed to
refute any effect of antibodies on cell viability. Following
incubation for 6 hours at 37.degree. C., cell viability was
evaluated using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) assay as described above.
[0212] Results
[0213] Lyophilized hIAPP was dissolved in 1,1,1,3,3,3
hexafluoro-2-propanol (HFIP) to eliminate any pre-existing
structures, after evaporation it was resuspended in 0.2 mM NaOH and
further diluted in phosphate buffer with 1% sodium dodecyl sulfate
(SDS). SDS is commonly-used as a chaotrophic agent; however, it can
be used to stabilize amyloid oligomers by producing a membrane like
environment.
[0214] In order to ascertain the exact nature of the assemblies
formed, the present inventors analyzed the resulting protein
species using several complementary methods. First, using PAGE
analysis under non-reducing conditions, the present inventors
observed three major assembly species: monomers of .about.3.9 kDa,
dimers of .about.8 kDa and trimers of .about.12 kDa (FIG. 1A). As a
negative control, rat IAPP peptide (rIAPP) was analyzed, which
differs by three residues and do not self assembles to form amyloid
structures.sup.30. Indeed, as expected, only the monomeric
conformation with rIAPP was observed (FIG. 1A). The oligomeric
structures, once assembled, were stable for up to seven days
without obvious disassembly or further polymerization to fibrils
(FIG. 1B).
[0215] In addition, the size distribution was analyzed under more
"native" conditions using fast liquid chromatography (FPLC)
performed under physiological conditions (PBS pH 7.4) (FIG. 1C).
Samples were loaded on a Superdex 75 HR10/300 column and their
molecular weight was determined using calibration curve. Using this
assay, the three species previously observed in the gel analysis
were observed together with an additional species of .about.90 kDa.
This conformer is probably not stable in SDS-PAGE and disassembles
to smaller multimers. Size exclusion chromatography did not show
evidence for larger assemblies which insinuates that amyloid
fibrils were not formed.
[0216] The morphology of the oligomers was examined by transmission
electron microscopy (TEM) and atomic force microscopy (AFM), (FIG.
1D). Using these methods only the .about.90 kDa oligomer could be
detected. These experiments revealed that no fibrillar aggregates
are present and the oligomers posses a spherical morphology with a
diameter of 5-30 nm as seen in TEM and a similar height (Z-axis)
dimension, which ranged between 8-35 nm, as measured by AFM.
Secondary structure determination was performed by circular
dichroism analysis (CD) in the far UV (200-240 nm) (FIG. 1E). CD
spectra indicated a predominant .alpha.-helical structure with two
negative peaks at 222 and 208 nm. The rIAPP treated in the same
manner exhibit lower .alpha.-helical content and higher
.beta.-sheet and random coil structures. Importantly, the CD
spectrum in analysis showed no evidence of .beta.-sheet structure
indicative of amyloid fiber formation. Several studies have shown
hIAPP adopts .alpha.-helical structure upon its interaction with
biological membranes.sup.27-28 suggesting an important role of this
conformation in the interaction with .beta.-cells. All these data
show that the present inventors were successful in their attempts
to stabilize soluble IAPP conformers. These oligomers were stable
and possessed unique physical properties which distinguish them
from mature amyloid structures.
[0217] Amyloid oligomers are considered toxic towards pancreatic
cells and cause membrane disruption. To examine whether the newly
formed conformers are indeed toxic, several complimentary methods
were used. As mentioned above, several studies have showed that
amyloid oligomers exhibits toxicity by forming discrete pores in
biological membranes. In order to examine whether hIAPP oligomers
act in the same manner, a liposomal system was used to model the
cell membrane. The liposomes were packed with fluorescent salt
(sodium calcein) and incubated with hIAPP oligomers (10 .mu.M,
37.degree. C.) or rIAPP as a negative control (FIG. 2B). Indeed,
hIAPP oligomers permeabilize the liposome's membrane rapidly
leading to the release of the fluorescent dye to the medium while
rIAPP did not exhibit any membrane damage abilities. These results
indicate that amyloid fiber formation is not necessary for membrane
disruption by hIAPP and by that imply a pivotal role of oligomers
as the toxic species. The toxicity of hIAPP oligomers towards cells
was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide-formazan assay (MTT assay) with pancreatic
RIN-m cells at different concentrations (FIG. 2A). Upon addition of
hIAPP oligomers (100 nM-10 .mu.M), MTT reduction was reduced,
indicating a dose dependent decrease in cell viability. The same
cells treated with rIAPP showed no such effect, again, pointing to
hIAPP oligomers as the primary toxic species.
[0218] To further study this cytotoxicity, fluorescence-activated
cell sorting (FACS) analysis was performed on pancreatic Rin-M cell
line (FIG. 2C). Cells treated with hIAPP oligomers were found to be
mostly in the early and late apoptotic phase. A clear and strong
correlation between oligomer concentration and the percentage of
apoptotic cells in culture could be observed (FIG. 2D). To
establish the physical interaction between oligomers and
.beta.-cells; hIAPP-Hiytelfluor 488 oligomers were incubated for
different periods of time and analyzed by confocal microscopy (FIG.
3). After one hour, an intensive localization of the oligomers to
the cell membrane was observed which was followed by insertion of
the oligomers to the cytoplasm, associated with a massive decline
in cell numbers. This was readily observed after four hours.
Dramatic cell morphology changes were observed after eight hours.
Once again these results emphasize the cytotoxic effect of the
oligomers. Thus, the effects of hIAPP oligomers were observed both
in vitro and ex-vivo. The present inventors wanted to obtain
further evidence regarding the relevance of these structures in a
more relevant physiological surrounding. In order to achieve this,
they examined whether they could identify specific antibodies that
recognize hIAPP assemblies from T2D patients. To this end, total
antibodies were purified from three T2D patients and three healthy
individuals and their ability to detect hIAPP oligomers was
compared. Dot-Blot analysis was used for quantitative assessment of
the purified serum antibodies affinity to the oligomers (FIG. 4A).
Antibodies from T2D patients exhibit strong recognition towards the
oligomers compared to purified antibodies from healthy people
serum. At 20 .mu.g/ml antibodies purified from T2D patients showed
higher binding activity than those purified from healthy
individuals, lower antibody concentrations showed only binding by
antibodies isolated from T2D patients (FIGS. 4A-4B).
[0219] In order to verify these results, the experiment was
repeated on a larger sample number. The present inventors purified
antibodies from 20 individuals, 10 T2D patients and 10 healthy
individuals (each group was comprised of 5 males and 5
females)--for patient details please see Table 2, herein below.
Every sample was examined for hIAPP oligomer recognition at 5
.mu.g/ml. Antibody recognition was quantitative assessed by 3,3',5,
5' tetramethyl benzidine (TMB) reagent. As illustrated in FIG. 4E,
T2D antibodies showed significant higher recognition properties
compared to purified antibodies from healthy people serum,
suggesting that T2D patients posses specific antibodies against
hIAPP assemblies.
TABLE-US-00002 TABLE 2 Lot Diagnosis Lot Number Age Gender
Medications Date Numbers Gender Age BRH436828 64 Male ActoplusMET,
Aug. 12, 2009 BRH436838 Male 22 ASA, Amlodipine, Benicar, Jalyn,
Lipitor, Naproxen BRH436829 43 Male ASA, Enalapril, October 2010
BRH436839 Male 32 Gabapentin, Glimipiride, Januvia, Levitra,
Metformin BRH436830 60 Male ASA, Aumentin, March 2003 BRH436840
Male 24 Benasepril- Hydrochlorothiazide, Cortisporin, Glimipiride,
Glucovance, Hydrocortisone, Metaxalone, Promethazine- DM,
Simvastatin, Tramadol, Zetia BRH436831 55 Male Enalapril, July 2006
BRH436841 Male 48 Flomax, Glucotrol XL, Glyburid, Lovastatin,
Metformin, Nexium, Simvastatin BRH436832 51 Male ASA, Diflucan,
October 2009 BRH436842 Male 24 Flonase, Glimepiride, Lantus,
Levitra, Lotrisone, Metformin, Zinthromax Z-Pak BRH436833 74 Female
Antivert, Benicar Dec. 8, 2010 BRH436843 Female 44 HCT, Coreg,
Glipizide, Janumet, Klor- Con M20, Lasix, Levaquin, Lidoderm,
Lotrisone, Metoprolol, Naproxen, Neurontin, Nexium, Nifedipine,
Pepcid, Ranexa, Robaxin, Simvastatin, Synthroid, Trazadone, Ultram
ER BRH436834 40 Female Actos, Anusol- February 2009 BRH436844
Female 37 HC, Coreg CR, Glucovance, Lotrel, Metformin BRH436835 48
Female Antivert, Byetta, 1998 BRH436845 Female 40 Enalapril,
Enalapril Maleate, Glyburide, Lantus, Lovastatin, Macrobid,
metformin, Metoprolol Tartrate, Vitamin D BRH436836 62 Female
ActoplusMET, January 2007 BRH436846 Female 47 Altace, Amaryl,
Avandia, Gemfibrozil, Lotrisone, Simvastatin BRH436837 69 Female
Actos, Ambien, April 2007 BRH436847 Female 28 Atenolol, Clonazepam,
Cozaar, Cymbalta, Ferrous Sulfate, Fosamax, Glucovan, Glyburide,
Januvia, Lexapro, Lisinopril, Lotrisone, Metformin, Monopril,
Nexium, Norvasc, Simvastatin
[0220] To show that the conformation that T2D patient antibodies
recognize is indeed the oligomeric one, PAGE analysis and
Western-blot analysis was used to study the hIAPP oligomers.
Positive control with commercial anti-IAPP antibody revealed
monomers, dimers and trimers assemblies: negative control of
purified antibodies from healthy people T2D antibodies did not
exhibit any binding activities. T2D patient antibodies specifically
recognized the oligomeric conformations but did not recognize the
monomeric form (FIG. 4D).
[0221] The present inventors further tested whether T2D-associated
antibodies can reduce the cytotoxic effect of hIAPP oligomers. In
vitro cellular viability experiments were performed with pancreatic
cells (FIG. 4C). As shown above, cell viability in the presence of
5 .mu.M oligomers was reduced dramatically as monitored by MTT
assay. In the presence of T2D antibodies, hIAPP oligomers toxicity
was reduced in a dose dependant manner. The same analysis was
preformed with non T2D antibodies and, as expected, these
antibodies did not exhibit any ability to reduce the cytotoxicity
of hIAPP oligomers.
[0222] The present results provide clear evidence for the role of
the oligomeric species, rather than the monomeric form of IAPP, in
the pathological cascade that result in cell death and loss of
pancreatic .beta.-cell mass. It is clear that the oligomeric
assemblies induce apoptotic cell death probably by their
interaction with the cell membrane. The ability of antibodies from
human patients to interact strongly and specifically with IAPP
oligomer clearly indicates that the formed assemblies as described
here represent valid epitopes present in diabetic patients.
Moreover, the ability of these antibodies to annul the toxic
activity of the oligomeric species paves the way for new
therapeutic approaches for the treatment of .beta.-cell mass loss
in the advanced stage of T2D. The newly indentified and
characterized species could serve as epitopes for the development
of immune response in active or passive immunization. Furthermore,
these species could serve as a platform for the screening and
optimization of small molecules that are able to interfere with the
toxic effect of oligomeric species.
[0223] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0224] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety 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. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
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Sequence CWU 1
1
1911992DNAHomo sapiens 1gggtatataa gagctggatt actagttagc aaatgagggg
gtaaatattc cagtggatac 60aagcttggac tcttttcttg aagctttctt tctatcagaa
gcatttgctg atattgctga 120cattgaaaca ttaaaagaaa atttgagaag
caatgggcat cctgaagctg caagtatttc 180tcattgtgct ctctgttgca
ttgaaccatc tgaaagctac acccattgaa agtcatcagg 240tggaaaagcg
gaaatgcaac actgccacat gtgcaacgca gcgcctggca aattttttag
300ttcattccag caacaacttt ggtgccattc tctcatctac caacgtggga
tccaatacat 360atggcaagag gaatgcagta gaggttttaa agagagagcc
actgaattac ttgccccttt 420agaggacaat gtaactctat agttattgtt
ttatgttcta gtgatttcct gtataattta 480acagtgccct tttcatctcc
agtgtgaata tatggtctgt gtgtctgatg tttgttgcta 540ggacatatac
cttctcaaaa gattgtttta tatgtagtac taactaaggt cccataataa
600aaagatagta tcttttaaaa tgaaatgttt ttgctataga tttgtatttt
aaaacataag 660aacgtcattt tgggacctat atctcagtgg cacaggttta
agaacgaagg agaaaaaggt 720agtttgaacc ttggtaaatt gtaaacagct
aataatgaag ttattcttga catgagaaaa 780tcagtaattg gaccaggcgc
ggtggctctt gcctgtaatc ccagcacttt gggaggccga 840ggcaggcaga
tcacaaggtc aggagttcga gaccagcctg accaacatgg tgaaaccctg
900tctctactaa aaatacaaaa attagccggg ggtggtgaca tgtgcctgta
atcccagcta 960ctcaggaggc taaggcagga gaatcgctta aacccaggag
gcggaggttg cagtgagccg 1020agattgcacc actgcactcc agcctgggtg
gcagagtgag actcgtctca aaaaaaagaa 1080agaaaattag taattgtaag
tacccctgat aagcaaatta gtaattgtca atacccctgt 1140taagcaattc
ctttttgcag tatatttctg aaatgacaga atgctgtttt aaaaacaaag
1200aaataaaatc ctgctcctga ctcggtcaaa atatttttta aagtctattg
tttgttgtgc 1260ttgctggtac taagaggcta tttaaaagta taaaactgct
ttgtatccat gagggtttca 1320ttgtgtgtta gcagcagtga gcttctatta
aatgtatatg tcatttattt tgtttaagtg 1380gctttcagca aacctcagtc
atattcttat gcagggtatt gcgaaacaac ttgtgttcta 1440ttaatcgtgt
cttcaattaa aagaccacag acttctggaa actctttgct gtataagaat
1500tatttctttt gtttaacaaa ttagacattt ctggcagagg ttatgtatat
gatacacttt 1560ttttgatagc agctgcaatg ttggacagaa gatgaaatgc
tttgctttga gtcagattct 1620tatgaatatc tgcttttccc tgactttgag
ttaggtagct ttggaagtag cattaattca 1680gataaactgc catcatgctg
cgttatgcca tttctaaaga cactcaactt gtacttttaa 1740aaaaatagaa
aaaataagca tttcaatcta agtggaaatt tgactcattg acttacattt
1800ctaagttaaa atttcccttt atgaagtgtg ccttaggtta ccaaattgta
gaggctttcg 1860ttggtggtgg taagtggtag cggtagtgag tgtatagagg
cagggaaata tatttataat 1920aaattctatg tcatgaatta catattgaaa
taaataggtg aatatacaaa tttataaaaa 1980aaaaaaaaaa aa 1992289PRTHomo
sapiens 2Met Gly Ile Leu Lys Leu Gln Val Phe Leu Ile Val Leu Ser
Val Ala 1 5 10 15 Leu Asn His Leu Lys Ala Thr Pro Ile Glu Ser His
Gln Val Glu Lys 20 25 30 Arg Lys Cys Asn Thr Ala Thr Cys Ala Thr
Gln Arg Leu Ala Asn Phe 35 40 45 Leu Val His Ser Ser Asn Asn Phe
Gly Ala Ile Leu Ser Ser Thr Asn 50 55 60 Val Gly Ser Asn Thr Tyr
Gly Lys Arg Asn Ala Val Glu Val Leu Lys 65 70 75 80 Arg Glu Pro Leu
Asn Tyr Leu Pro Leu 85 3725DNAMus musculus 3gtcgtgcatg cagacttggg
ctgtagttcc tgaagcttca ggctgtcaaa gcattttctg 60atattgctgc ctcggaccac
tgaaagggat cttgagaaat gatgtgcatc tccaaactgc 120cagctgtcct
cctcatcctc tctgtggcac tgaaccactt gagagctaca cctgtcagaa
180gtggtagcaa ccctcagatg gacaaacgga agtgcaacac ggccacgtgt
gccacacaac 240gcctggcaaa ctttttggtt cgttccagca acaaccttgg
tccagtcctc ccaccaacca 300acgtgggatc gaatacatat ggcaagagga
atgcggcagg ggatccaaat agggaatcct 360tggatttctt actcgtttaa
agtcaatgta cttctgcagc acttaatact ttatgtgtaa 420atgctctggt
gatttcctga atattaacag tacctttttc attcccccct cagtgagaat
480gcacaatgtg cttgtgcttg atgactgtgt gtgtaaattc tcatgctaag
aattgcttta 540aactgagtat tgatcaagtt cagagtgaag tcaatgtctc
taatcacaca tgttcttgct 600atacatttat attttaggga cacttaaaat
ttctgttttt accttgtacc tctatgactc 660aagtttaaca ataaagaaga
ccatgggatg atgaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaa 725493PRTMus
musculus 4Met Met Cys Ile Ser Lys Leu Pro Ala Val Leu Leu Ile Leu
Ser Val 1 5 10 15 Ala Leu Asn His Leu Arg Ala Thr Pro Val Arg Ser
Gly Ser Asn Pro 20 25 30 Gln Met Asp Lys Arg Lys Cys Asn Thr Ala
Thr Cys Ala Thr Gln Arg 35 40 45 Leu Ala Asn Phe Leu Val Arg Ser
Ser Asn Asn Leu Gly Pro Val Leu 50 55 60 Pro Pro Thr Asn Val Gly
Ser Asn Thr Tyr Gly Lys Arg Asn Ala Ala 65 70 75 80 Gly Asp Pro Asn
Arg Glu Ser Leu Asp Phe Leu Leu Val 85 90 5238PRTAequorea
macrodactyla 5Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Ile Val Pro
Val Leu Ile 1 5 10 15 Glu Leu Asp Gly Asp Val His Gly His Lys Phe
Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Asp Tyr Gly Lys
Leu Glu Ile Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val
Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Gly Tyr Gly Ile Gln
Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met 65 70 75 80 Asn Asp Phe
Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg 85 90 95 Thr
Ile Phe Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val 100 105
110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Met
115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Met Pro Asp Lys
Ala Asn Asn Gly 145 150 155 160 Leu Lys Val Asn Phe Lys Ile Arg His
Asn Ile Glu Gly Gly Gly Val 165 170 175 Gln Leu Ala Asp His Tyr Gln
Thr Asn Val Pro Leu Gly Asp Gly Pro 180 185 190 Val Leu Ile Pro Ile
Asn His Tyr Leu Ser Leu Gln Thr Ala Ile Ser 195 200 205 Lys Asp Arg
Asn Glu Thr Arg Asp His Met Val Phe Leu Glu Phe Phe 210 215 220 Ser
Ala Cys Gly His Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235
6717DNAaequorea macrodactyla 6atgagtaaag gagaagaact tttcactggg
attgtcccag ttctcattga gttagacggt 60gatgtccatg gacataaatt ctctgtcaga
ggagaagggg aaggcgatgc agattatgga 120aaacttgaaa tcaaattcat
ttgcactact ggaaagctac cagttccatg gccaacactt 180gttactacac
tgggctacgg catccaatgt ttcgcaagat acccagaaca catgaaaatg
240aatgacttct tcaagagtgc catgcctgag ggttacattc aagaaagaac
catctttttc 300caagatgatg gaaaatacaa gacacgtggt gaagtcaagt
ttgaaggtga tactcttgtt 360aacagaattg agctcaaagg tatggacttt
aaagaagatg gcaatatcct tggacacaag 420ttggagtaca attttaattc
acataatgta tacattatgc cggacaaagc caataatgga 480ctcaaagtca
atttcaaaat tagacacaat atcgaaggtg gtggtgtcca acttgctgat
540cattaccaaa caaatgttcc ccttggagac ggtcctgtcc ttataccaat
caatcactac 600ctatccttgc aaacagccat ttcaaaagat cgaaatgaga
cgagagatca tatggtgttt 660ctggaatttt tctcagcttg tggacataca
catggcatgg atgaactata caaataa 7177489PRTArtificial sequenceAlkaline
phosphatase 7Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu
Leu Phe Thr 1 5 10 15 Pro Val Thr Lys Ala Arg Thr Pro Glu Met Pro
Leu Gln Gly Thr Ala 20 25 30 Val Asp Gly Gly Gly Gly Ser Met His
Ala Ser Leu Glu Val Leu Glu 35 40 45 Asn Arg Ala Ala Gln Gly Asp
Ile Thr Ala Pro Gly Gly Ala Arg Arg 50 55 60 Leu Thr Gly Asp Gln
Thr Ala Ala Leu Arg Asp Ser Leu Ser Asp Lys 65 70 75 80 Pro Ala Lys
Asn Ile Ile Leu Leu Ile Gly Asp Gly Met Gly Asp Ser 85 90 95 Glu
Ile Thr Ala Ala Arg Asn Tyr Ala Glu Gly Ala Gly Gly Phe Phe 100 105
110 Lys Gly Ile Asp Ala Leu Pro Leu Thr Gly Gln Tyr Thr His Tyr Ala
115 120 125 Leu Asn Lys Lys Thr Gly Lys Pro Asp Tyr Val Thr Asp Ser
Ala Ala 130 135 140 Ser Ala Thr Ala Trp Ser Thr Gly Val Lys Thr Tyr
Asn Gly Ala Leu 145 150 155 160 Gly Val Asp Ile His Glu Lys Asp His
Pro Thr Ile Leu Glu Met Ala 165 170 175 Lys Ala Ala Gly Leu Ala Thr
Gly Asn Val Ser Thr Ala Glu Leu Gln 180 185 190 Asp Ala Thr Pro Ala
Ala Leu Val Ala His Val Thr Ser Arg Lys Cys 195 200 205 Tyr Gly Pro
Ser Ala Thr Ser Glu Lys Cys Pro Gly Asn Ala Leu Glu 210 215 220 Lys
Gly Gly Lys Gly Ser Ile Thr Glu Gln Leu Leu Asn Ala Arg Ala 225 230
235 240 Asp Val Thr Leu Gly Gly Gly Ala Lys Thr Phe Ala Glu Thr Ala
Thr 245 250 255 Ala Gly Glu Trp Gln Gly Lys Thr Leu Arg Glu Gln Ala
Gln Ala Arg 260 265 270 Gly Tyr Gln Leu Val Ser Asp Ala Ala Ser Leu
Asn Ser Val Thr Glu 275 280 285 Ala Asn Gln Gln Lys Pro Leu Leu Gly
Leu Phe Ala Asp Gly Asn Met 290 295 300 Pro Val Arg Trp Leu Gly Pro
Lys Ala Thr Tyr His Gly Asn Ile Asp 305 310 315 320 Lys Pro Ala Val
Thr Cys Thr Pro Asn Pro Gln Arg Asn Asp Ser Val 325 330 335 Pro Thr
Leu Ala Gln Met Thr Asp Lys Ala Ile Glu Leu Leu Ser Lys 340 345 350
Asn Glu Lys Gly Phe Phe Leu Gln Val Glu Gly Ala Ser Ile Asp Lys 355
360 365 Gln Asp His Ala Ala Asn Pro Cys Gly Gln Ile Gly Glu Thr Val
Asp 370 375 380 Leu Asp Glu Ala Val Gln Arg Ala Leu Glu Phe Ala Lys
Lys Glu Gly 385 390 395 400 Asn Thr Leu Val Ile Val Thr Ala Asp His
Ala His Ala Ser Gln Ile 405 410 415 Val Ala Pro Asp Thr Lys Ala Pro
Gly Leu Thr Gln Ala Leu Asn Thr 420 425 430 Lys Asp Gly Ala Val Met
Val Met Ser Tyr Gly Asn Ser Glu Glu Asp 435 440 445 Ser Gln Glu His
Thr Gly Ser Gln Leu Arg Ile Ala Ala Tyr Gly Pro 450 455 460 His Ala
Ala Asn Val Val Gly Leu Thr Asp Gln Thr Asp Leu Phe Tyr 465 470 475
480 Thr Met Lys Ala Ala Leu Gly Leu Lys 485 81470DNAArtificial
sequenceAlkaline phosphatase coding sequence 8ttatttcagc cccagagcgg
ctttcatggt gtagaagaga tcggtctggt cggtcagtcc 60aacaacattg gcggcatgcg
ggccatacgc cgcaatacgc aactgactgc cggtatgttc 120ttgtgaatcc
tcttcggagt tcccgtaact catcaccatc actgcgccat ctttggtatt
180tagcgcctgg gtgaggcccg gagctttggt atccggcgca acaatctggc
tggcgtgggc 240gtgatcagcg gtgactatga ccagcgtgtt accctccttt
ttagcgaatt ccagcgcccg 300ttgtacggct tcatcgagat cgaccgtctc
gccaatttgc ccacaaggat tcgcagcatg 360atcctgttta tcgattgacg
caccttcaac ttgcaggaaa aagcctttct catttttact 420caacaattca
atggctttgt cggtcatctg cgccagggtt ggtacactgt cattacgttg
480cggatttggc gtacaggtga ctgcgggctt atcgatattg ccatggtacg
ttgctttcgg 540tcctagccag cgcactggca tattgccgtc agcaaacagg
ccaagcaggg gtttttgctg 600attcgcttcc gtcaccgaat tcagtgaggc
agcatcgctc accaactgat aaccacgcgc 660ctgtgcctgt tcacgcagcg
tttttccctg ccattcacca gcggttgccg tttcagcaaa 720ggtttttgcg
ccgccgccaa gcgtaacgtc ggcacgagcg ttaagcagct gttcggtaat
780cgatcctttt ccgccttttt ccagagcgtt acccggacat ttttcactgg
tcgcgctcgg 840accgtagcat ttgcgcgagg tcacatgtgc caccagcgca
gcgggcgtgg catcctgcaa 900ctctgcggta gaaacgttac cggtcgccag
acctgcggct tttgccattt ccagaatcgt 960tgggtgatct ttttcgtgaa
tatcgacgcc cagcgcgccg ttataggttt tgacaccggt 1020tgaccaggcg
gttgctgatg cagccgagtc ggtgacgtag tccggtttgc cggttttttt
1080attcagcgca tagtgagtgt attgcccggt aagcggtaag gcatctatac
ctttaaaaaa 1140gccgcccgca ccttcggcat aattacgtgc ggcagtaatt
tccgagtccc ccatcccatc 1200gccaatcagc aaaataatat tttttgcagg
tttatcgcta agagaatcac gcagagcggc 1260agtctgatca cccgttaaac
ggcgagcacc gccgggtgca gtaatatcgc cctgagcagc 1320ccggttttcc
agaacctcga ggctagcatg catagaaccg ccaccaccgt cgacagcggt
1380accctgcaga ggcatttctg gtgtccgggc ttttgtcaca ggggtaaaca
gtaacggtaa 1440gagtgccagt gcaatagtgc tttgtttcac
14709309PRTArtificial sequencePeroxidase 9Met Gln Leu Thr Pro Thr
Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn Ile Val Arg
Asp Thr Ile Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 Ile Ala
Ala Ser Ile Leu Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45
Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr 50
55 60 Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Pro
Val 65 70 75 80 Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro
Arg Thr Val 85 90 95 Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln
Gln Ser Val Thr Leu 100 105 110 Ala Gly Gly Pro Ser Trp Arg Val Pro
Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Ala Phe Leu Asp Leu Ala
Asn Ala Asn Leu Pro Ala Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu
Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser
Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175
Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180
185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg
Gly 195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp
Phe Asp Leu 210 215 220 Arg Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr
Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu Ile Gln Ser Asp
Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Ala Thr Asp Thr Ile Pro
Leu Val Arg Ser Phe Ala Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn
Ala Phe Val Glu Ala Met Asp Arg Met Gly Asn Ile 275 280 285 Thr Pro
Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu Asn Cys Arg Val 290 295 300
Val Asn Ser Asn Ser 305 10955DNAArtificial sequencePeroxidase
coding sequence 10aagcttaacc atgcagttaa cccctacatt ctacgacaat
agctgtccca acgtgtccaa 60catcgttcgc gacacaatcg tcaacgagct cagatccgat
cccaggatcg ctgcttcaat 120attacgtctg cacttccatg actgcttcgt
gaatggttgc gacgctagca tattactgga 180caacaccacc agtttccgca
ctgaaaagga tgcattcggg aacgctaaca gcgccagggg 240ctttccagtg
atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag
300ttgtgcagac ctgctgacta tagctgcgca acagagcgtg actcttgcag
gcggaccgtc 360ctggagagtg ccgctcggtc gacgtgactc cctacaggca
ttcctagatc tggccaacgc 420caacttgcct gctccattct tcaccctgcc
ccagctgaag gatagcttta gaaacgtggg 480tctgaatcgc tcgagtgacc
ttgtggctct gtccggagga cacacatttg gaaagaacca 540gtgtaggttc
atcatggata ggctctacaa tttcagcaac actgggttac ctgaccccac
600gctgaacact acgtatctcc agacactgag aggcttgtgc ccactgaatg
gcaacctcag 660tgcactagtg gactttgatc tgcggacccc aaccatcttc
gataacaagt actatgtgaa 720tctagaggag cagaaaggcc tgatacagag
tgatcaagaa ctgtttagca gtccaaacgc 780cactgacacc atcccactgg
tgagaagttt tgctaactct actcaaacct tctttaacgc 840cttcgtggaa
gccatggacc gtatgggtaa cattacccct ctgacgggta cccaaggcca
900gattcgtctg aactgcagag tggtcaacag caactcttaa taaggatccg aattc
95511286PRTArtificial sequenceSynthetic construct translated
protein product 11Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly
Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser
Ser Asp Ile Gly Thr Tyr 20 25 30 Lys Ile Val Ser Trp Tyr Gln Gln
His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Asn
Gln Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys
Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95
Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser 100
105
110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala Leu
115 120 125 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 130 135 140 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe Thr Ser Tyr 145 150 155 160 Trp Ile Gly Trp Val Arg Gln Met Pro
Gly Lys Gly Leu Glu Trp Met 165 170 175 Gly Ile Ile Tyr Pro Gly Asp
Ser Asp Thr Arg Tyr Ser Pro Ser Phe 180 185 190 Gln Gly Gln Val Thr
Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 195 200 205 Leu Gln Trp
Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 210 215 220 Ala
Arg His Arg Ala Ala Ser Gly Ser Pro Asp Ala Cys Asp Tyr Trp 225 230
235 240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala
Pro 245 250 255 Thr Leu Phe Pro Ala Ala Ala His His His His His His
Gly Ala Ala 260 265 270 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
Gly Ala Ala 275 280 285 12861DNAArtificial sequenceSynthetic
construct 12cagtctgtgt tgacgcagcc gccctcagtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgatattggg acttataaaa ttgtctcctg
gtaccaacag 120caccctggca aagcccccaa actcatgatt tatgacgtca
atcagcggcc ctcaggggtt 180tctgatcgct tctctggctc caagtctggc
aacacggcct ccctgacaat ctctgggctc 240caggctgagg acgaggctga
ttattactgc agctcatata caagcggcag cactctggta 300ttcggcgggg
ggaccaagct gaccgtccta ggctcgagtg gtggaggcgg ttcaggcgga
360ggtggctctg gcggtagtgc acttcaggta cagctgcagc agtcaggagc
agaggtgaaa 420aagcccgggg agtctctgaa gatctcctgt aagggttctg
gatacagctt taccagctac 480tggatcggct gggtgcgcca gatgcccggg
aaaggcctgg agtggatggg gatcatctat 540cctggtgact ctgataccag
atacagcccg tccttccaag gccaggtcac catctcagcc 600gacaagtcca
tcagcaccgc ctacctgcag tggagcagcc tgaaggcctc ggacaccgcc
660atgtattact gtgcgagaca tcgggccgct agtgggagcc cggacgcgtg
tgactactgg 720ggccagggaa ccctggtcac cgtctcctca gggagtgcat
ccgccccaac ccttttcccc 780gcggccgcac atcatcatca ccatcacggg
gccgcagaac aaaaactcat ctcagaagag 840gatctgaatg gggccgcata g
8611315PRTArtificial sequenceBiotin lygase tag 13Leu His His Ile
Leu Asp Ala Gln Lys Met Val Trp Asn His Arg 1 5 10 15
14238PRTArtificial sequenceOrange fluorescent protein 14Met Ser Lys
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu
Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu 20 25
30 Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys
35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
Thr Leu 50 55 60 Gly Tyr Gly Ile Leu Cys Phe Ala Arg Tyr Pro Glu
His Met Lys Met 65 70 75 80 Asn Asp Phe Phe Lys Ser Ala Met Pro Glu
Gly Tyr Ile Gln Glu Arg 85 90 95 Thr Ile Phe Phe Gln Asp Asp Gly
Lys Tyr Lys Thr Arg Gly Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr
Leu Val Asn Arg Ile Glu Leu Lys Gly Met 115 120 125 Asp Phe Lys Glu
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn
Ser His Asn Val Tyr Ile Met Pro Asp Lys Ala Asn Asn Gly 145 150 155
160 Leu Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Gly Gly Gly Val
165 170 175 Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp
Gly Pro 180 185 190 Val Leu Ile Pro Ile Asn His Tyr Leu Ser Tyr Gln
Thr Ala Ile Ser 195 200 205 Lys Asp Arg Asn Glu Thr Arg Asp His Met
Val Phe Leu Glu Phe Phe 210 215 220 Ser Ala Cys Gly His Thr His Gly
Met Asp Glu Leu Tyr Lys 225 230 235 15717DNAArtificial
sequenceOrange fluorescent protein coding sequence 15atgagtaaag
gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60gatgtccatg
gacataaatt ctctgtcaga ggagaagggg aaggcgatgc agattatgga
120aaacttgaaa tcaaattcat ttgcactact ggaaagctac cagttccatg
gccaacactt 180gttactacac tgggctatgg catcctatgt ttcgcaagat
acccagaaca catgaaaatg 240aatgacttct tcaagagtgc catgcctgag
ggttacattc aagaaagaac catctttttc 300caagatgatg gaaaatacaa
gacacgtggt gaagtcaagt ttgaaggtga tactcttgtt 360aacagaattg
agctcaaagg tatggacttt aaagaagatg gcaatatcct tggacacaag
420ttggagtaca attttaactc acataatgta tacattatgc cggacaaagc
caataatgga 480ctcaaagtca atttcaaaat tagacacaat atcgaaggtg
gtggtgtcca actcgctgat 540cattaccaaa caaatgttcc ccttggagac
ggtcctgtcc ttataccaat caatcactac 600ctatcctatc aaacagccat
ttcaaaagat cgaaatgaga cgagagatca tatggtgttt 660ctggaatttt
tctcagcttg tggacataca catggcatgg atgaactata caaataa
717161019PRTArtificial sequenceBeta-galactosidase 16Met Ala Asp Pro
Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly 1 5 10 15 Val Thr
Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp 20 25 30
Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg 35
40 45 Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu
Ala 50 55 60 Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu Ala
Asp Thr Val 65 70 75 80 Val Val Pro Ser Asn Trp Gln Met His Gly Tyr
Asp Ala Pro Ile Tyr 85 90 95 Thr Asn Val Thr Tyr Pro Ile Thr Val
Asn Pro Pro Phe Val Pro Thr 100 105 110 Glu Asn Pro Thr Gly Cys Tyr
Ser Leu Thr Phe Asn Val Asp Glu Ser 115 120 125 Trp Leu Gln Glu Gly
Gln Thr Arg Ile Ile Phe Asp Gly Val Asn Ser 130 135 140 Ala Phe His
Leu Trp Cys Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp 145 150 155 160
Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly 165
170 175 Glu Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser Asp Gly Ser
Tyr 180 185 190 Leu Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile Phe
Arg Asp Val 195 200 205 Ser Leu Leu His Lys Pro Thr Thr Gln Ile Ser
Asp Phe His Val Ala 210 215 220 Thr Arg Phe Asn Asp Asp Phe Ser Arg
Ala Val Leu Glu Ala Glu Val 225 230 235 240 Gln Met Cys Gly Glu Leu
Arg Asp Tyr Leu Arg Val Thr Val Ser Leu 245 250 255 Trp Gln Gly Glu
Thr Gln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly 260 265 270 Glu Ile
Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg 275 280 285
Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu 290
295 300 Tyr Arg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile
Glu 305 310 315 320 Ala Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg
Ile Glu Asn Gly 325 330 335 Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu
Ile Arg Gly Val Asn Arg 340 345 350 His Glu His His Pro Leu His Gly
Gln Val Met Asp Glu Gln Thr Met 355 360 365 Val Gln Asp Ile Leu Leu
Met Lys Gln Asn Asn Phe Asn Ala Val Arg 370 375 380 Cys Ser His Tyr
Pro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg 385 390 395 400 Tyr
Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu Thr His Gly Met 405 410
415 Val Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met
420 425 430 Ser Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn His
Pro Ser 435 440 445 Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His
Gly Ala Asn His 450 455 460 Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val
Asp Pro Ser Arg Pro Val 465 470 475 480 Gln Tyr Glu Gly Gly Gly Ala
Asp Thr Thr Ala Thr Asp Ile Ile Cys 485 490 495 Pro Met Tyr Ala Arg
Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro 500 505 510 Lys Trp Ser
Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro 515 520 525 Leu
Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly 530 535
540 Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly
545 550 555 560 Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile Lys
Tyr Asp Glu 565 570 575 Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp
Phe Gly Asp Thr Pro 580 585 590 Asn Asp Arg Gln Phe Cys Met Asn Gly
Leu Val Phe Ala Asp Arg Thr 595 600 605 Pro His Pro Ala Leu Thr Glu
Ala Lys His Gln Gln Gln Phe Phe Gln 610 615 620 Phe Arg Leu Ser Gly
Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe 625 630 635 640 Arg His
Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp Gly 645 650 655
Lys Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly 660
665 670 Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala
Gly 675 680 685 Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala
Thr Ala Trp 690 695 700 Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln
Trp Arg Leu Ala Glu 705 710 715 720 Asn Leu Ser Val Thr Leu Pro Ala
Ala Ser His Ala Ile Pro His Leu 725 730 735 Thr Thr Ser Glu Met Asp
Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp 740 745 750 Gln Phe Asn Arg
Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp 755 760 765 Lys Lys
Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro 770 775 780
Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn 785
790 795 800 Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr Gln Ala
Glu Ala 805 810 815 Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp
Ala Val Leu Ile 820 825 830 Thr Thr Ala His Ala Trp Gln His Gln Gly
Lys Thr Leu Phe Ile Ser 835 840 845 Arg Lys Thr Tyr Arg Ile Asp Gly
Ser Gly Gln Met Ala Ile Thr Val 850 855 860 Asp Val Glu Val Ala Ser
Asp Thr Pro His Pro Ala Arg Ile Gly Leu 865 870 875 880 Asn Cys Gln
Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly Leu 885 890 895 Gly
Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp 900 905
910 Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro
915 920 925 Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr
Gly Pro 930 935 940 His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser
Arg Tyr Ser Gln 945 950 955 960 Gln Gln Leu Met Glu Thr Ser His Arg
His Leu Leu His Ala Glu Glu 965 970 975 Gly Thr Trp Leu Asn Ile Asp
Gly Phe His Met Gly Ile Gly Gly Asp 980 985 990 Asp Ser Trp Ser Pro
Ser Val Ser Ala Asp Phe Gln Leu Ser Ala Gly 995 1000 1005 Arg Tyr
His Tyr Gln Leu Val Trp Cys Gln Lys 1010 1015 173060DNAArtificial
sequenceBeta-galactosidase coding sequene 17ttatttttga caccagacca
actggtaatg gtagcgaccg gcgctcagct ggaaatccgc 60cgatactgac gggctccagg
agtcgtcgcc accaatcccc atatggaaac cgtcgatatt 120cagccatgtg
ccttcttccg cgtgcagcag atggcgatgg ctggtttcca tcagttgctg
180ttgactgtag cggctgatgt tgaactggaa gtcgccgcgc cactggtgtg
ggccataatt 240caattcgcgc gtcccgcagc gcagaccgtt ttcgctcggg
aagacgtacg gggtatacat 300gtctgacaat ggcagatccc agcggtcaaa
acaggcggca gtaaggcggt cgggatagtt 360ttcttgcggc cctaatccga
gccagtttac ccgctctgct acctgcgcca gctggcagtt 420caggccaatc
cgcgccggat gcggtgtatc gctcgccact tcaacatcaa cggtaatcgc
480catttgacca ctaccatcaa tccggtaggt tttccggctg ataaataagg
ttttcccctg 540atgctgccac gcgtgagcgg tcgtaatcag caccgcatca
gcaagtgtat ctgccgtgca 600ctgcaacaac gctgcttcgg cctggtaatg
gcccgccgcc ttccagcgtt cgacccaggc 660gttagggtca atgcgggtcg
cttcacttac gccaatgtcg ttatccagcg gtgcacgggt 720gaactgatcg
cgcagcggcg tcagcagttg ttttttatcg ccaatccaca tctgtgaaag
780aaagcctgac tggcggttaa attgccaacg cttattaccc agctcgatgc
aaaaatccat 840ttcgctggtg gtcagatgcg ggatggcgtg ggacgcggcg
gggagcgtca cactgaggtt 900ttccgccaga cgccactgct gccaggcgct
gatgtgcccg gcttctgacc atgcggtcgc 960gttcggttgc actacgcgta
ctgtgagcca gagttgcccg gcgctctccg gctgcggtag 1020ttcaggcagt
tcaatcaact gtttaccttg tggagcgaca tccagaggca cttcaccgct
1080tgccagcggc ttaccatcca gcgccaccat ccagtgcagg agctcgttat
cgctatgacg 1140gaacaggtat tcgctggtca cttcgatggt ttgcccggat
aaacggaact ggaaaaactg 1200ctgctggtgt tttgcttccg tcagcgctgg
atgcggcgtg cggtcggcaa agaccagacc 1260gttcatacag aactggcgat
cgttcggcgt atcgccaaaa tcaccgccgt aagccgacca 1320cgggttgccg
ttttcatcat atttaatcag cgactgatcc acccagtccc agacgaagcc
1380gccctgtaaa cggggatact gacgaaacgc ctgccagtat ttagcgaaac
cgccaagact 1440gttacccatc gcgtgggcgt attcgcaaag gatcagcggg
cgcgtctctc caggtagcga 1500aagccatttt ttgatggacc atttcggcac
agccgggaag ggctggtctt catccacgcg 1560cgcgtacatc gggcaaataa
tatcggtggc cgtggtgtcg gctccgccgc cttcatactg 1620caccgggcgg
gaaggatcga cagatttgat ccagcgatac agcgcgtcgt gattagcgcc
1680gtggcctgat tcattcccca gcgaccagat gatcacactc gggtgattac
gatcgcgctg 1740caccattcgc gttacgcgtt cgctcatcgc cggtagccag
cgcggatcat cggtcagacg 1800attcattggc accatgccgt gggtttcaat
attggcttca tccaccacat acaggccgta 1860gcggtcgcac agcgtgtacc
acagcggatg gttcggataa tgcgaacagc gcacggcgtt 1920aaagttgttc
tgcttcatca gcaggatatc ctgcaccatc gtctgctcat ccatgacctg
1980accatgcaga ggatgatgct cgtgacggtt aacgcctcga atcagcaacg
gcttgccgtt 2040cagcagcagc agaccatttt caatccgcac ctcgcggaaa
ccgacatcgc aggcttctgc 2100ttcaatcagc gtgccgtcgg cggtgtgcag
ttcaaccacc gcacgataga gattcgggat 2160ttcggcgctc cacagtttcg
ggttttcgac gttcagacgt agtgtgacgc gatcggcata 2220accaccacgc
tcatcgataa tttcaccgcc gaaaggcgcg gtgccgctgg cgacctgcgt
2280ttcaccctgc cataaagaaa ctgttacccg taggtagtca cgcaactcgc
cgcacatctg 2340aacttcagcc tccagtacag cgcggctgaa atcatcatta
aagcgagtgg caacatggaa 2400atcgctgatt tgtgtagtcg gtttatgcag
caacgagacg tcacggaaaa tgccgctcat 2460ccgccacata tcctgatctt
ccagataact gccgtcactc caacgcagca ccatcaccgc 2520gaggcggttt
tctccggcgc gtaaaaatgc gctcaggtca aattcagacg gcaaacgact
2580gtcctggccg taaccgaccc agcgcccgtt gcaccacaga tgaaacgccg
agttaacgcc 2640atcaaaaata attcgcgtct ggccttcctg tagccagctt
tcatcaacat taaatgtgag 2700cgagtaacaa cccgtcggat tctccgtggg
aacaaacggc ggattgaccg taatgggata 2760ggttacgttg gtgtagatgg
gcgcatcgta accgtgcatc tgccagtttg aggggacgac 2820gacagtatcg
gcctcaggaa gatcgcactc cagccagctt tccggcaccg cttctggtgc
2880cggaaaccag gcaaagcgcc attcgccatt caggctgcgc aactgttggg
aagggcgatc 2940ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt 3000aagttgggta acgccagggt tttcccagtc
acgacgttgt aaaacgacgg gatcagccat 306018159PRTArtificial
sequencestreptavidin 18Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser
Ala Ala Glu Ala Gly 1 5 10 15 Ile Thr Gly Thr Trp Tyr Asn Gln Leu
Gly Ser Thr Phe Ile Val Thr 20 25 30 Ala Gly Ala Asp Gly Ala Leu
Thr Gly Thr Tyr Glu Ser Ala Val Gly 35 40 45 Asn Ala Glu Ser Arg
Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro 50 55 60 Ala Thr Asp
Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys 65 70 75 80 Asn
Asn Tyr Arg Asn Ala His Ser Ala Thr Thr
Trp Ser Gly Gln Tyr 85 90 95 Val Gly Gly Ala Glu Ala Arg Ile Asn
Thr Gln Trp Leu Leu Thr Ser 100 105 110 Gly Thr Thr Glu Ala Asn Ala
Trp Lys Ser Thr Leu Val Gly His Asp 115 120 125 Thr Phe Thr Lys Val
Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys 130 135 140 Lys Ala Gly
Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln 145 150 155
19483DNAArtificial sequencestreptavidin coding sequence
19gacccgagca aagattctaa agcacaagta tctgctgcag aagcaggaat tacaggcaca
60tggtataatc agctgggatc tacatttatt gttacagccg gcgcagatgg agctcttaca
120ggaacatatg aatctgctgt tggaaatgca gaatctagat acgtgcttac
aggaagatat 180gattctgcac ctgcaacaga tggatccgga acagcacttg
gatggacagt tgcatggaaa 240aacaattata gaaacgcaca tagcgctaca
acatggtctg gccaatatgt gggaggtgca 300gaagcaagaa ttaacacaca
atggctttta acatctggaa caacagaagc aaatgcatgg 360aaaagtactc
ttgttggaca tgatacattt acaaaagtta aacctagcgc agcatctatc
420gatgcagcga aaaaagcagg agttaacaat ggcaatcctt tagatgcagt
tcaacaataa 480tga 483
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