U.S. patent application number 17/087614 was filed with the patent office on 2021-05-06 for composition for improving skin conditions comprising a fragment of human heat shock protein 90a as an active ingredient.
The applicant listed for this patent is REGERON, INC.. Invention is credited to Youngwook CHO, Kyunyoung LEE, Kibum NAM, Dahlkyun OH.
Application Number | 20210130423 17/087614 |
Document ID | / |
Family ID | 1000005332986 |
Filed Date | 2021-05-06 |
![](/patent/app/20210130423/US20210130423A1-20210506\US20210130423A1-2021050)
United States Patent
Application |
20210130423 |
Kind Code |
A1 |
NAM; Kibum ; et al. |
May 6, 2021 |
COMPOSITION FOR IMPROVING SKIN CONDITIONS COMPRISING A FRAGMENT OF
HUMAN HEAT SHOCK PROTEIN 90A AS AN ACTIVE INGREDIENT
Abstract
Liposomal and/or nano-liposomal encapsulated peptides of HSP90a,
HPf polypeptide (115 aa) and novel polypeptides HPf.DELTA.C1 (101
aa) and HPf.DELTA.C2 (87 aa), and methods for
manufacturing/preparing and using the compositions, are disclosed.
Chimeric fusion proteins that include HSP90a, HPf, HPfAC, HPfAC2
polypeptide, or combinations thereof, are presented. Transformed
cell lines and expression vectors capable of expressing the
chimeric fusion proteins, are provided. Methods for producing large
amounts of recombinant HSP90a, HPf polypeptide, HPf.DELTA.C1 or
HPf.DELTA.C2 polypeptide, using expression vectors and transformed
cell lines, are described. Topical and other delivery form
preparations, including microneedle preparations, and methods for
using the preparations for improving skin conditions (atopic
dermatitis, wrinkles, skin elasticity, dark spots (over
pigmentation), overall skin rejuvenation, skin ageing) and other
therapeutic (anti-cancer, anti-ALS, anti-Huntington's disease,
obesity) and cosmeceutical uses are presented. Wound healing
preparations with the Hsp90a and related peptides are
disclosed.
Inventors: |
NAM; Kibum; (Chuncheon-si,
KR) ; LEE; Kyunyoung; (Chuncheon-si, KR) ;
CHO; Youngwook; (Chuncheon-si, KR) ; OH;
Dahlkyun; (Chuncheon-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGERON, INC. |
Chuncheon-sh |
|
KR |
|
|
Family ID: |
1000005332986 |
Appl. No.: |
17/087614 |
Filed: |
November 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15952100 |
Apr 12, 2018 |
10822382 |
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17087614 |
|
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14911215 |
Jun 20, 2016 |
9956263 |
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PCT/KR2014/007430 |
Aug 11, 2014 |
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15952100 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0014 20130101;
A61Q 19/08 20130101; C07K 2319/24 20130101; C07K 14/47 20130101;
C07K 2319/35 20130101; A61M 2037/0023 20130101; A61K 9/06 20130101;
A61K 47/10 20130101; A61Q 19/02 20130101; A61P 17/00 20180101; A61Q
19/00 20130101; A61M 37/0015 20130101; A61L 26/0066 20130101; A61K
9/0021 20130101; A61P 25/28 20180101; A61L 2300/252 20130101; C07K
14/245 20130101; A61K 47/32 20130101; C07K 14/61 20130101; A61K
9/127 20130101; A61K 8/027 20130101; A61K 8/64 20130101; A61M
2037/0061 20130101; A61K 38/1709 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61L 26/00 20060101 A61L026/00; A61P 25/28 20060101
A61P025/28; A61P 17/00 20060101 A61P017/00; A61K 9/00 20060101
A61K009/00; C07K 14/245 20060101 C07K014/245; A61K 8/64 20060101
A61K008/64; A61K 47/10 20060101 A61K047/10; A61K 9/127 20060101
A61K009/127; A61Q 19/02 20060101 A61Q019/02; A61Q 19/08 20060101
A61Q019/08; A61M 37/00 20060101 A61M037/00; A61K 9/06 20060101
A61K009/06; C07K 14/61 20060101 C07K014/61; A61Q 19/00 20060101
A61Q019/00; A61K 8/02 20060101 A61K008/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2013 |
KR |
10-2013-0094930 |
Claims
1. A chimeric construct encoding a fusion protein comprising a
nucleic acid sequence encoding HSP90a, HPf polypeptide,
HFf.DELTA.C1 or HPf.DELTA.C2 polypeptide, or a fragment thereof,
and a nucleic acid sequence encoding a fusion partner peptide.
2. The chimeric construct of claim 1 wherein the fusion partner
peptide is theoredoxin A, maltose binding protein (MBP) or human
growth hormone (hGH).
3. The chimeric construct of claim 2 further comprising a protein
cleavage enzyme recognition site located between the nucleic acid
sequence encoding the HPf peptide and the nucleic acid sequence
encoding the fusion partner peptide.
4. The chimeric construct of claim 1 wherein the HPf polypeptide
has a nucleic acid sequence of SEQ ID. NO. 1.
5. A transformed cell line transformed to express a HPf-fusion
partner chimeric protein, said cell line comprising a TOP 10 cell
line, an RZ4500 cell line, a BL21(DE3)pLyS cell line or a
RosettaBlue (DE3) cell line.
6. The transformed cell line of claim 5 wherein the HPf-fusion
partner chimeric protein is TRX(TEVc)-HPf fusion protein,
MBP(TEVc)-HPf fusion protein, or TRX(NGc)-HPf fusion protein.
7. A method for preparing a nano-liposomal encapsulated HSP90a,
HPf, HFf.DELTA.C1 or HPf.DELTA.C2 polypeptide composition, said
method comprising: (a) dissolving a phospholipid capable of forming
a liposome with an HSP90a, HPf, HFf.DELTA.C1 or HPf.DELTA.C2
polypeptide, in a buffered aqueous solution of salt, said
phospholipid comprising yellow yolk lecithin or soybean lecithin;
and (b) passing the aqueous solution through a high-pressure
homogenizer while gradually increasing the content of the
phospholipid and the pressure of the high-pressure homogenizer as
the number of the passages increases to provide an HSP90a, HPf,
HFf.DELTA.C1 or HPf.DELTA.C2 polypeptide-containing nano-liposomal
encapsulated HPf polypeptide composition.
8. The method of claim 7 wherein the polypeptide is HSP90a.
9. A method for treating a skin condition, wherein the skin
condition is atopic dermatitis, wrinkles, dark spots, skin
elasticity or skin aging, comprising: providing a composition
comprising a concentration of about 100 ng/ml to about 1 mg/ml of
an Hsp90a, HPf, HFf.DELTA.C1 or HPf.DELTA.C2 polypeptide, or a
combination thereof, to a skin area demonstrating atopic
dermatitis, wrinkles, dark spots, skin elasticity or skin aging;
and reducing the skin condition at the skin area.
10. A method for reducing subcutaneous fat accumulation comprising:
providing a composition comprising a concentration of about 100
ng/ml to about 1 mg/ml of an Hsp90a, HPf, HFf.DELTA.C1,
HPf.DELTA.C2 polypeptide, or a combination thereof, to an area
presenting subcutaneous fat accumulation; and reducing subcutaneous
fat in the area on which the composition is applied.
11. The method of claim 10 wherein the composition comprises a
patch or body wrap including a microneedle formulation of the
Hsp90a, HPf, HFf.DELTA.C1, HPf.DELTA.C2, or a combination
thereof.
12. A method for inhibiting synuclein-induced toxicity related
neurological disease, comprising: providing a composition
comprising a concentration of about 100 ng/ml to about 1 mg/ml of
an HPf polypeptide, HPf polypeptide, HFf.DELTA.C1, HPf.DELTA.C2, or
fragment thereof, to a patient having a synuclein-induced toxicity
related neurological disease; and inhibiting neurological
degeneration from synuclein-induced toxicity.
13. The method of claim 12 wherein the neurological disease is
Huntington's disease or Amyotrophic lateral sclerosis.
14. A dissolvable microneedle formulation comprising HSP90a, HPf
polypeptide, HPf.DELTA.C1, HPf.DELTA.C2, or a combination
thereof.
15. The dissolvable microneedle formulation of claim 14 wherein the
HPf polypeptide has a sequence of SEQ ID NO 1, the HPf.DELTA.C1 has
a sequence of SEQ ID NO 20 and the HPf.DELTA.C2 has a sequence of
SEQ ID NO 21.
16. The dissolvable microneedle formulation of claim 14 comprising
a patch, body wrap or facial mask.
17. A method for treating a neurodegenerative disease associated
with neurotoxic tau accumulation comprising: providing a patient
having a neurodegenerative disease with a therapeutic agent
comprising a peptide of HSP90a, HPf polypeptide, HPf.DELTA.C1,
HPf.DELTA.C2, or a combination thereof; and reducing neurotoxic tau
species accumulation, wherein generation of neurotoxic tau species
is inhibited.
18. The method of claim 17 wherein the therapeutic agent comprises
a fusion peptide, wherein said fusion peptide comprises a peptide
of HSP90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2, or a combination
thereof and a growth factor peptide or protein.
19. The method of claim 18 wherein the growth factor is human
growth factor.
20. The method of claim 17 wherein the neurodegenerative disease is
Alzheimers disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
15/952,100, filed Apr. 12, 2018, now U.S. Pat. No. 10,822,382,
which is a continuation-in-part patent application of U.S. patent
application Ser. No. 14/911,215, filed Jun. 20, 2016, now U.S. Pat.
No. 9,956,263. U.S. Ser. No. 14/911,215 is a United States national
stage entry patent application of International PCT Application
PCT/KR2014/007430 filed with the Republic of Korea Receiving Office
on Aug. 11, 2014. The PCT application claims the benefit of
priority to Republic of Korea patent application 10-2013-0094930,
filed on Aug. 9, 2013.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 22, 2021, is named 119522-000035 v1_SL.txt and is 24,462
bytes in size.
BACKGROUND OF THE INVENTION
Technical Field
[0003] The present invention relates to compositions suitable for
topical administration, the compositions comprising
pharmacologically active polypeptides that are encapsulated in a
liposome and/or nano-liposome. The invention also relates to
methods of manufacturing the liposome and/or nano-liposome
formulations. The invention further relates to methods for
improving and/or treating skin conditions, enhancing wound healing,
and for inhibiting subcutaneous fat formation.
Related Art
[0004] Heat Shock Protein 90a, abbreviated as HSP90a hereafter, is
a dimer composed of two monomers containing phosphate groups, and
having a molecular weight of 90 KD. The two monomers tend to become
easily oligomerized under some conditions, e.g., when present in
aqueous solution. Most Heat Shock Proteins have been known to
function intracellularly. Other reports indicate that some Heat
Shock Proteins work outside the cell, suggesting alternative
physiological roles. The role of HSP90a in immune-regulation has
been suggested.sup.10,12. However, no systemic studies have been
carried out with HSP90a, nor has it been described as having any
activity for affecting skin conditions, such as atopic dermatitis
or skin aging, or as affecting subcutaneous fat formation or
accumulation. The relatively large size of the HSP90a fragment has
precluded the use of this molecule in topical preparations, as it
is unable to penetrate to the skin dermis layer.sup.6.
[0005] Atopic dermatitis (AD) is a chronic dermal disorder caused
by defects in stratum corneum, which is generally considered
idiopathic.sup.9. It affects children and adults as well. Its
epidemiology has been known to associate with hereditary.sup.4 or
environmental causes.sup.7, and immunological factors.sup.9.
[0006] There is no known cure for AD thus far, although treatments
may reduce the severity and frequency of flares. Commonly used
compositions for treating atopic dermatitis include small molecule
based compounds with properties of anti-histamine, steroids or
immune suppression. Alternatively systemic immune suppressing
agents may be tried such as cyclosporine, methotrexate, interferon
gamma-1b, mycophenolate mofetil and azathioprine.sup.4. However
since these small-molecules based compounds accompany such serious
adverse effects as deterioration of immune function upon long term
use, new materials to overcome such barriers are needed in the
treatment of these and other conditions.
[0007] Unlike small molecule based medicine, there are significant
advantages in using a polypeptide as active ingredients for the
treatment of dermal disorders and/or preparing skin cosmetic
products. For example, polypeptides are generally more compatible
with interactions with the immune system and cells, and generally
decomposed in a pro-physiological manner within the body, hence
generating fewer side effects compared to small molecule (chemical)
containing preparations, especially during long term use.
Furthermore, as relates to uses in cosmeceutical preparations,
small molecule containing cosmetic products generally produce only
short term cosmetic effects, while polypeptide containing
cosmeceutical preparations have been described as providing longer
term improvement of overall skin condition, and even skin
rejuvenation.sup.3. However, the overall size and bulkiness of many
potentially useful polypeptides prevents the penetration of these
ingredients into skin tissues.
[0008] Traditionally, macromolecules having a molecular weight of
500 Daltons or more are considered too large to pass through the
skin epidermis due to the skin keratin barrier.sup.6. Even when
used with chemical penetration enhancers, macromolecules having a
molecular weight of more than 2000 Daltons are considered
practically implausible for topical use, as they are unable to
penetrate the skin epidermis. Therefore, peptides developed as
pharmaceutical/cosmetic ingredients have been limited to those
having a much smaller size, such as a size of less than 10 amino
acids (roughly about 1100 Daltons MW), so as to optimize the
delivery of the active ingredient to the skin dermis. Thus, many
potentially useful polypeptides having a size of 10 amino acids or
greater have not been utilized in topical preparations. Delivery of
an active ingredient, such as a polypeptide, to the skin dermis
layer, is necessary to provide the most pharmaceutically meaningful
outcomes with functional pharmaceutical/cosmetic preparations.
[0009] Liposome based delivery of human growth hormone (hGH),
having a MW of 22,124 Daltons (191 amino acid size), has been
reported.sup.1-3. However, challenges associated with effective
topical delivery of other pharmacologically different
peptides/proteins, such as heat shock protein Hsp90a, remain.
[0010] A 115 amino acid fragment of Heat Shock Protein, termed HPf,
is encoded by an amino acid sequence spanning between the linker
and the middle domain of the native endogenous HSP sequence (FIG.
1). This fragment has been reported to ameliorate skin necrosis
caused by diabetic ulcer..sup.8 Improvements in delivery products
are, however, lacking for facilitating fuller use and formulation
of these and related polypeptides.
[0011] Subcutaneous fat is the layer of subcutaneous tissue that is
most widely distributed and is mainly composed of adipocytes. The
number of adipocytes varies among different areas of the body,
while their size varies according to the body's nutritional state
(Subcutaneous Tissue, Medical Subject Headings (MeSH), NLM
Retrieved 5 Jun. 2013). Some reports suggest that reducing the size
of fat cells could improve fat cell sensitivity to insulin.sup.5.
Numerous small molecule based oral delivery medicines have been
developed and marketed for suppressing the accumulation of fat.
Oral administration of these types of preparations, however, is
associated with adverse side effects. A topical preparation would
be more effective in such applications, and would offer the
advantage of targeting problem fat deposit areas on the body, among
other advantages.
[0012] One of the many barriers in the use of polypeptides in
topical preparations remains the size and bulkiness of these
polypeptide and protein molecules, which, because of the structure
of skin tissues, do not penetrate the skin sufficiently to provide
beneficial pharmacological and physiological effects in the body.
Conventional approaches to this problem have been the use of
mesotherapeutic devices, such as micro needles, electroporation
devices, laser treatments, and infrared irradiation. For a variety
of reasons, these approaches have not provided a sufficiently
effective and convenient approach for topical administration of
peptide-containing preparations. Problems associated with
sufficient shelf-life and product biological stability also limit
the use of polypeptide/peptide/protein based topical and other
preparations.
[0013] A need continues to exist in the medical arts for improved
topical preparations with preserved bioactivity and enhanced
shelf-life of identified polypeptide/protein-based molecules. In
addition, a need continues to exist for achieving effective
delivery of these and other potent polypeptide/protein agents deep
into skin tissues to achieve maximal physiological benefit to the
patient. The present invention provides a solution to these and
other technical problems in the medical arts for the use of
polypeptide and/or protein-based molecules in topical and other
delivery formulation applications and treatment methods.
SUMMARY OF THE INVENTION
[0014] The present invention provides, for the first time,
liposomal and nano-liposomal encapsulated Heat Shock Protein (HSP)
preparations, as well as preparations that include smaller
polypeptide fragments of HSP, namely HPf (115 aa), as well as novel
polypeptides HPf.DELTA.C1 (101 aa), and HPf.DELTA.C2 (87 aa). The
liposomal preparations are further demonstrated to possess a number
of novel and advantageous physiological effects when delivered
topically at the skin surface, including the enhancement of wound
healing, the inhibition of fat cell differentiation, the
improvement of skin conditions (including atopic dermatitis,
wrinkle, skin elasticity and dark spots, and promoting overall skin
rejuvenation) and effective delivery to skin hair follicles.
[0015] The polypeptide compositions and preparations may further be
provided as nano-liposomal encapsulated preparations. These
preparations are demonstrated to possess long term storage
stability and retained bioactivity in solution. The preparations
may be provided in a delivery form suitable for topical,
mesotherapeutic or systemic administration.
[0016] Surprisingly, the present invention has accomplished the
effective delivery of HPf, a 115 amino acid fragment of HSP90a, to
the stratum corneum of both intact skin and wounded skin, using a
topical formulation of the polypeptide in a liposome-based delivery
preparation.
[0017] According to some aspects of the invention, a liposomal
(particularly, a nano-liposomal) encapsulated polypeptide
composition is provided comprising a Heat Shock Protein, and HPf
polypeptide or fragment thereof, as an active ingredient. The HPf
polypeptide fragment may comprise a polypeptide having a 115 aa
sequence (termed HPf) (SEQ ID. No. 1), a 101 aa sequence
(HPf.DELTA.C1) (SEQ ID. NO. 20), an 87 aa sequence (HPf.DELTA.C2)
(SEQ ID. NO. 21), an HSP90a aa sequence (SEQ. ID NO. 2), or a
combination thereof. The composition, in some embodiments, is
formulated so as to be suitable for topical application to the
skin, and in particular, for use in the preparation of
cosmeceutical preparations, (cosmetics, skin conditioners, and the
like).
[0018] In particular embodiments, the nano-liposomes have a
particle size of 50-500 nm, 50-350 nm, or 100-250 nm.
[0019] The present invention includes the discovery that HSP90a
fragments, such as HPf, as well as synthetic polypeptide sequences
that are unlike the native sequence, such as HPf.DELTA.C1 (101 aa),
and HPf.DELTA.C2 (87 aa), promote the differentiation of the skin
cells, both epidermal and dermal, while inhibiting the
differentiation of preadipocytes at the subdermal layer. This
activity, in turn, inhibits the progression and severity of atopic
eczema and/or atopic dermatitis. This feature provides yet another
objective of the present invention.
[0020] In another embodiment of the present invention, a
composition is provided for use in a medicament for suppressing
subcutaneous fat accumulation and fat cell differentiation.
[0021] In another aspect, the invention provides a method for
reducing and/or inhibiting the accumulation of subcutaneous fat
and/or suppressing subcutaneous fat cell differentiation is
provided, the method comprising topically applying a nano-liposomal
composition comprising a polypeptide having a sequence
corresponding to a fragment of Heat Shock Protein. In some
embodiments, the polypeptide is defined by a 115 aa sequence
(termed HPf) (SEQ ID. No. 1), a 101 aa sequence (HPf.DELTA.C1) (SEQ
ID. NO. 20), an 87 aa sequence (HPf.DELTA.C2) (SEQ ID. NO. 21), or
an HSP90a aa sequence (SEQ. ID NO. 2), the polypeptide being
encapsulated in a nano-liposome.
[0022] In another aspect, the invention provides a nano-liposomal
preparation for use in a medicament for treatment of obesity,
cellulite, varicose veins of lower extremities with ulcer, lower
body extremity edema, varicose veins, skin discoloration, venous
eczema, scleroderma, inflammatory thrombus, skin ulcer, or chronic
pain.
[0023] Yet another aspect of the invention provides for transformed
cell lines useful in the production and/or manufacture of
recombinant HSP90a and HPf polypeptides (HSP90a, HPf, HPf.DELTA.C1,
HPf.DELTA.C2). By way of example, cell lines that may be used in
the preparation of these transformed cell lines include a TOP 10
cell line, a BL21(D3)pLys cell line, RosettaBlue(DE3) cell line,
and RZ4500 cell line. Expression vectors that include a sequence
encoding a fusion protein comprising the HSP90a HPf, and/or HPf
polypeptide fragments, with a fusion partner protein/peptide, are
also disclosed, and are useful in the large-scale and economical
production of these useful therapeutic polypeptides. The fusion
protein constructs are also defined as part of the present
invention.
[0024] Another aspect of the invention provides for a method of
manufacturing recombinant HSP90a and HPf polypeptides, including
the HSP90a, HPf, HPf.DELTA.C1, and HPf.DELTA.C2 polypeptides.
[0025] Yet another aspect of the invention provides a topical
liposomal polypeptide formulation containing HSP90a, an HPf
polypeptide (HSP90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2), or a
combination thereof, for use in the treatment of a skin condition,
wherein the skin condition is atopic dermatitis, wrinkles, dark
spots, skin elasticity or skin aging, wherein said composition
comprises a concentration of about 100 ng/ml to about 1 mg/ml of
the HPf polypeptide or HPf polypeptide fragment.
[0026] Yet another aspect of the invention provides a topical
liposomal polypeptide formulation containing HSP90a, an HPf
polypeptide (HSP90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2), or a
combination thereof, for use in the treatment of subcutaneous fat
accumulation, wherein said formulation comprises a concentration of
about 100 ng/ml to about 1 mg/ml of the polypeptide.
[0027] The invention also provides for a use of a HSP90a, an HPf
polypeptide or fragment thereof (HPf.DELTA.C1, HPf.DELTA.C2), or a
combination thereof, in the manufacture of a preparation for the
treatment of a skin condition, wherein the skin condition is atopic
dermatitis, wrinkles, dark spots, skin elasticity or skin
aging.
[0028] The invention also provides for a use of a HSP90a, an HPf
polypeptide or fragment thereof (HPf.DELTA.C1, HPf.DELTA.C2), or a
combination thereof in the manufacture of a preparation for the
treatment of obesity, cellulite, varicose veins of lower
extremities with ulcer, lower body extremity edema, varicose veins,
skin discoloration, venous eczema, scleroderma, inflammatory
thrombus, skin ulcer, or chronic pain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows the sequence of three (3) fragments of the
endogenous HSP90a polypeptide: a 115 amino acid fragment, (Glu236aa
to Asp350aa), named HPf; a 101 amino acid fragment (Glu236-Glu336),
named HPf.DELTA.C1; and an 87 amino acid fragment (Glu236-Asp 322),
named HPf.DELTA.C2. HPf and HPf.DELTA.C2 are used as active
ingredients of the present preparations/formulations.
[0030] FIG. 2-1A shows the recombinant fusion protein constructs of
HPf, and the fusion partner thioredoxin A (TRX), TRX(NGc)-HPf (FIG.
2-1B) and TRX(TEVc)-HPf(FIG. 2-1C), with the hydroxylamine and TEV
protease recognition site respectively inserted in between the two,
which is needed for facile cleavage and purification of HPf. FIG.
2-2A illustrates the structures of HPf.DELTA.C1,
TRX(TEVc)-HPf.DELTA.C1 (FIG. 2-2B), HPf.DELTA.C2 (FIG. 2-2C) and
TRX(TEVc)-HPf.DELTA.C2 (FIG. 2-2D). The TEV protease recognition
site was inserted after TRX, which is coupled with HPf.DELTA.C1 or
HPf.DELTA.C2, in order to facilitate cleavage of the fusion
proteins and purification of HPf.DELTA.C1 or HPf.DELTA.C2. FIG.
2-3A shows the recombinant fusion protein construct of the fusion
partner maltose binding protein (MBP) and TEV including a
His.times.6 ("His.times.6" disclosed as SEQ ID NO: 34),
MBP(TEVc)-His-TEV, and the recombinant fusion construct of MBP and
HPf without the His.times.6 ("His.times.6" disclosed as SEQ ID NO:
34), MBP(TEVc)-HPf (FIG. 2-3B). The TEV protease recognition site
inserted in between each fusion construct is needed for facile
cleavage and purification of TEV or HPf. FIG. 2-4 shows the
recombinant fusion protein construct of HPf and the fusion partner
human growth hormone (HGH), HGH(TEVc)-HPf, with the TEV protease
recognition site inserted in between the two, which is needed for
facile cleavage and purification of HPf. FIG. 2-5B shows the
locations of the primers used for cloning the HSP90a gene, FIG.
2-5A shows the results of the PCR products amplified by said
primers.
[0031] FIG. 3-1A and FIG. 3-1B are the result of the SDS-PAGE of
the recombinant proteins HPf, TRX(NGc)-HPf, and TRX(TEVc)-HPf
produced by expression of their recombinant expression vectors. The
recombinant expression vector constructs were expressed in the
RZ4500, BL21(DE3)pLyS, and RosettaBlue(DE3) cell lines to quantify
the expression levels of these expression vector constructs. FIG.
3-2A is the result of the SDS-PAGE of the small-scale (5 ml)
protein expression experiments relating to HPf.DELTA.C2. FIG. 3-2B
relates to TRX(TEVc)-HPf.DELTA.C1. FIG. 3-2C relates to
TRX(TEVc)-HPf.DELTA.C2. FIG. 3-3 is the result of the SDS-PAGE of
the recombinant protein MBP(TEVc)-HPf produced by expression of its
recombinant expression vector. FIG. 3-4A and FIG. 3-4B are the
result of the SDS-PAGE of the recombinant protein HGH(TEVc)-HPf
produced by expression of its recombinant expression vector. FIG.
3-5 is the result of the SDS-PAGE of the HSP90a protein, produced
by E. coli cells transformed by the HSP90a expression vector.
[0032] FIG. 4A is the gel results of HPf peptide production with
TRX(TEVc)-HPf fusion protein (1. Control, 2. HPf, 3. Control, 4.
TRX(TEVc)-HPf). 4B is the gel results of HPf peptide production
with HGH(TEVc)-HPf fusion construct. (1. HPf, 2. HGH(TEVc)-HPf, 3.
Full HSP90a protein.
[0033] FIG. 5A shows the change of TRX(TEVc)-HPf fusion protein
production with increasing culture time in a large scale
fermentation (50 liter) for preparing the protein: 5B shows change
in dissolved oxygen. 5C shows change in pH (5C), and 5D shows
change in optical density.
[0034] FIG. 6 demonstrates results of the isolation of HPf from the
recombinant TRX(TEVc)-HPf fusion protein, separation as an
inclusion body, and its TEV-cleavage efficiency depending on the
amount of TEV added. 1. Protein marker, 2. Competent cell (negative
control), 3. Total cell lysate, 4. Supernatant fraction after
homogenization, 5. Pellet after sonication (Inclusion body), 6.
Washing solution of the pellet, 7. Solubilized inclusion body.
8-11. Solubilized inclusion body+TEV protease.
[0035] FIG. 7A is the result of the HPLC, and FIG. 7B is the
SDS-PAGE, confirming the purity of the purified HPf.
[0036] FIG. 8 describes the MALDI-TOF analysis results of the HPf
protein confirming its aa sequence identity with HSP90a.
[0037] FIG. 9-1A is the ELS and GFC analysis results of purified
HPf1 estimating masses, sizes, and numbers of different HPf1
aggregates formed during its purification. demonstrates the Ls int.
Distribution (IS); FIG. 9-1B demonstrates the Wt. conv.
Distribution (WT); FIG. 9-1C demonstrates the No cony. Distribution
(NO); FIG. 9-1D demonstrates the GFC (Gel Filtration
Chromatography) profile. FIG. 9-2 is a particle size analysis of
HPf using TEM electron micrographs (EF-TEM; Energy
Filtering-Transmission Electron Microscope, KBSI, Korea).
[0038] FIG. 10A-1 shows the effect of varying HPf concentration on
24-hour incubation survival rate of an keratinocyte cell-line
(HaCaT) and FIGS. 10A-2, 10B-1, 10B-2, and 10B-3 show the effects
of varying HPf concentrations on 24, 48, 120, and 168 hour
incubation survival rates of embryonic fibroblast cells (HEF),
respectively.
[0039] FIG. 11A shows the stability of HPf protein kept in a gel
state while varying the temperature and storage time. FIG. 11B
shows the stability of the HPf protein kept in a buffer solution
state while varying the temperature and storage time.
[0040] FIG. 12 demonstrates the ability of HPf to inhibit the
degranulation in RBL-2H3 cell line as measured by the activity of
secreting beta-hexosaminidase.
[0041] FIG. 13A shows the time line and HPf treatments examined.
FIG. 13 B-1 shows the condition of atopic dermatitis with no DNFB.
FIG. 13B-2 shows the condition of atopic dermatitis with DNFB only,
FIG. 13B-3 shows the condition of atopic dermatitis with
DNFB+control, and FIG. 13 B-4 dhows the condition of atopic
dermatitis improved by topical administration of HPf on wounds
induced by applying DNFB on the NC/Nga mouse skin.
[0042] FIG. 14A-1 demonstrates changes in the skin tissue structure
with no treatment, 14A-2 with DNFB only treatment, 14A-3 with
DNFB+Control-1, and 14A-4 with DNFB+HPf-1 treatment; FIGS. 14B-1
through 14B-4 show simply the same results with a different
corresponding set of hystological specimens. HPf was applied
topically on wounds induced by applying DNFB on the NC/Nga mouse
skin.
[0043] FIG. 15A shows the expression level of KRT 10 (Keratin 10),
TGM 1 (Transglutaminase 1) or IVL (involucrin) genes in an
epidermal cell line (HaCaT) (a keratinocyte). FIG. 15B shows the
expression level of KRT10, TGM 1 or IVL genes in a dermal cell line
(CCD986-sk) (a fibroblast) treated with HPf or PBS (control). In
the course of skin epidermal stem cell differentiation,
keratinocytes increase the expression of genes related to Keratin
10, Transglutaminase 1 and involucrin. Thus, the KRT 10, TGM 1 or
IVL genes are used as markers that reflect the degree of cell
differentiation in keratinocyte cells.
[0044] FIG. 16A shows the effects of HPf on the subcutaneous fat
cell differentiation confirmed by Red 0 stain and FIG. 16B presents
a graphical representation of FIG. 16A data.
[0045] FIG. 17A (C-1, C-2, C-3)--Control; FIG. 17B (H-1, H-2, H-3,
H-4)--topical HPf application to artificial human skin. FIG.
17C--graph showing HPf topical application promotes thickening of
the dermis layer in the structure of artificial human skin.
[0046] FIG. 18A--Cell viability after application of 100 .mu.g/ml
HPf. HPf does not affect cell viability; FIG. 18B--Melanin Content
after application of 100 .mu.g ml HPf. HPf inhibits melanin
biosynthesis.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present inventors have performed intensive research in
the identification and manufacture of topical liposomal
encapsulated HPf polypeptide and HPf polypeptide fragment
compositions having potent pharmacological activity in vivo. The
topical preparations include a polypeptide encoded by the amino
acid at SEQ ID. No. 1, or a fragment thereof, as an active
ingredient. The pharmacological activity of the compositions
include improvement of skin conditions, including atopic
dermatitis, wrinkles, dark spots, improving skin elasticity and
skin rejuvenation, as well as enhancing wound healing. In addition,
the compositions are also demonstrated to inhibit subcutaneous fat
cell differentiation and to suppress the accumulation of
subcutaneous fat.
[0048] The term `human heat shock protein 90a fragment` or `HSP90a
fragment` represents the HSP90a of which partial sequences were
removed by biochemical or DNA recombinant techniques. A polypeptide
fragment of HSP90a is described as HPf herein. HPf is a 115 amino
acid fragment of endogenous HSP90a, and is encoded by the sequence
spanning from amino acid (aa) 236 to aa 350, including the "Linker"
region (see FIG. 1). HPf.DELTA.C1 is the 101 amino acid fragment of
the endogenous HSP90a encoded by the sequence spanning from aa 236
to aa 336; and HPf.DELTA.C2 is the 87 amino acid fragment of the
endogenous HSP90a encoded by the sequence spanning from aa236 to aa
322 (see FIG. 1) of the full amino acid sequence of HSP90a (SEQ ID.
NO. 2).
[0049] As HPf protein showed a very high propensity of forming
aggregates as characterized by ELS and GFC analyses of FIG. 9, its
aa sequence and 3D structure were examined for the reasons of HPf
aggregation, and the present inventors sought to devise ways to
overcome this aggregation problem. The present inventors suspected
a hydrophobic stretch of aa sequence in HPf might be the reason for
this aggregation. Therefore, two other constructs were designed,
HPf.DELTA.C1 and HPf.DELTA.C2, that eliminated the hydrophobic
stretch of HPf, and presented novel polypeptides. The resultant
HPf.DELTA.C1 and HPf.DELTA.C2 showed much better aggregation
profile, and hence gave HPf.DELTA.C1 or HPf.DELTA.C2 separation and
purification advantages over HPf. HPf.DELTA.C1 construct gave a
soluble HPf.DELTA.C1 protein form while HPf.DELTA.C2 gave an
inclusion body form when each over-expression was attempted. To
increase the separation yield and facilitate the purification
efficiency, the smallest fragment HPf.DELTA.C2 was chosen for
further studies. The HPf.DELTA.C2 polypeptide was surprisingly
found to be at least as active as HPf, and in some parameters, to
be even more active than HPf.
[0050] The biochemical/biological properties of the HPf and
HPf.DELTA.C2 can be determined based on the following three
factors: 1) Over 90% of the amino acid sequence identity with HPf
or HPf.DELTA.C2, 2) Binding of each fragment to the receptor or
other binding proteins of the endogenous HSP90a, and 3) the
biological activity of HPf or HPf.DELTA.C2.
[0051] According to some embodiments, a composition according to
the present invention is a phospholipid or liposome composition,
and preferably a liposome or nano-liposomal composition. In some
embodiments, the HPf (encoded by SEQ ID. NO. 1) is encapsulated in
liposomes or nano-liposomes, and applied to the skin. According to
some embodiments, the inventive composition is a nano-liposomal
composition formulated for topical administration.
[0052] As used herein, the term "nano-liposome" refers to a
liposome having the form of conventional liposome and a mean
particle diameter of 20-1000 nm. According to some embodiments, the
mean particle diameter of the nano-liposome is 50-500 nm, more
preferably 50-350 nm, and most preferably 100-250 nm.
[0053] As used herein, the term "liposome" refers to a spherical
phospholipid vesicle of colloidal particles which are associated
with themselves, and liposomes composed of amphiphilic molecules,
each having a water soluble head (hydrophilic group) and a water
insoluble tail (hydrophobic group), and show a structure aligned by
spontaneous binding caused by the interaction there between. The
liposome is classified, according to the size and lamellarity
thereof, into SUV (small unilamellar vesicle), LUV (large
unilamellar vesicle) and MLV (multi lamellar vesicle). The
liposomes showing various lamellarities as described above have a
double membrane structure similar to the cell membrane.
[0054] The nano-liposome and liposome of the present invention can
be prepared using phospholipid, polyol, a surfactant, fatty acid,
salt and/or water.
[0055] The phospholipid which is a component used in the
preparation of the liposome and nano-liposome, is used as an
amphipathic lipid. By way of example, such amphipathic lipids
include natural phospholipids (e.g., egg yolk lecithin, soybean
lecithin, and sphingomyelin) and synthetic phospholipids (e.g.,
dipalmitoylphosphatidyl-choline or hydrogenated lecithin), the
lecithin being preferred. More preferably, the lecithin is a
naturally derived unsaturated or saturated lecithin extracted from
soybean or egg yolk.
[0056] Polyols which can be used in the preparation of the
inventive nano-liposome are not specifically limited, and may
include propylene glycol, dipropylene glycol, 1,3-butylene glycol,
glycerin, methylpropanediol, isoprene glycol, pentylene glycol,
erythritol, xylitol and sorbitol.
[0057] The surfactant which can be used in the preparation of the
inventive nano-liposome may be any surfactant known in the art, and
examples thereof include anionic surfactants (e.g., alkyl acyl
glutamate, alkyl phosphate, alkyl lactate, dialkyl phosphate and
trialkyl phosphate), cationic surfactants, amphoteric surfactants
and nonionic surfactants (e.g., alkoxylated alkylether, alkoxylated
alkylester, alkylpolyglycoside, polyglycerylester and sugar
ester).
[0058] The fatty acids which can be used in the preparation of the
inventive nano-liposome are higher fatty acids, and preferably
saturated or unsaturated fatty acid having a CI 2-22 alkyl chain,
and examples thereof include lauric acid, myristic acid, palmitic
acid, stearic acid, oleic acid and linoleic acid.
[0059] Water which is used in the preparation of the inventive
nano-liposome is generally deionized distilled water.
[0060] According to some embodiments, the inventive nano-liposome
is prepared only with phospholipid, salt and water, as described in
detail in the Examples below.
[0061] According to some embodiments, the HPf-containing
nano-liposome is prepared through a process comprising the steps
of: (a) dissolving a phospholipid capable of forming liposome
(preferably, yellow yolk lecithin or soybean lecithin) in a
buffered aqueous solution of salt containing HPf; and (b) passing
the aqueous solution containing HPf and phospholipid through a
high-pressure homogenizer while gradually increasing the content of
the phospholipid and the pressure of the high-pressure homogenizer
as the number of the passages increases, thus preparing a
HPf-containing nano-liposome.
[0062] The aqueous solution containing HPf is preferably a buffer
solution having a pH of 6-8, and more preferably about 7, for
example, sodium phosphate buffer solution. If the sodium phosphate
buffer solution is used, the concentration thereof will preferably
be 5-100 mM, more preferably 5-60 mM, even more preferably 10-30
mM, and most preferably about 20 mM.
[0063] The mixture of the phospholipid and the HPf-containing
aqueous solution is passed through a high-pressure homogenizer
several times, in which the amount of the phospholipid and the
pressure of the homogenizer are gradually increased as the number
of the passages increases. According to a preferred embodiment of
the present invention, the pressure of the homogenizer is increased
gradually to 0-1000 bar, and preferably 0-800 bar. The pressure can
be increased by 50 bar or 100 bar in each cycle, and preferably 100
bar. According to a preferred embodiment of the present invention,
the amount of the phospholipid is gradually increased to 5-40 w/v
(%) in each cycle, and more preferably 5-30 w/v (%). Through the
high-pressure homogenization process including these gradual
increases in phospholipid content and pressure, an HPf-containing
nano-liposome is prepared and a liquid HPf-containing nano-liposome
is preferably prepared.
[0064] The present invention is shown herein to be effective for
treating atopic dermatitis. While not wishing to be limited to any
particular theory or mechanism of action, it is contemplated that
this effect may be the result of suppressing the immune function
around the affected areas while simultaneously healing the wounds,
whereas anti-histamine or steroid containing compositions
traditionally used for atopic dermatitis work only by suppressing
the immune functions without a wound healing activity.
[0065] The composition of the present invention is also shown to
provide an improvement of various other skin conditions. For
example, the compositions provide an effective treatment for
various skin conditions, including wrinkles, dark spots, improving
skin elasticity, reducing skin aging, and improving skin
moisture.
[0066] Furthermore, the composition of the present invention is
effective in suppressing the subcutaneous fat cell differentiation
hence reducing the subcutaneous fat accumulation. Accordingly, the
liposome encapsulated HPf of the present invention is effective for
treating obesity, and the accompanying adversities, such as
cellulite, varicose veins of lower extremities with ulcer, the
edema of lower extremities due to the varicose veins, coloration of
the skin, venous eczema, scleroderma, inflammatory thrombus, skin
ulcer, chronic pain, disablement of leg functions or any
combination of the above symptoms due to the obesity.
[0067] The present composition may be provided as a cosmetic or
pharmaceutical composition. Accordingly, the active and effective
ingredients include compositions that are commonly used for
preparing cosmetic products, such as a stabilizer, emulsifier,
vitamins, coloring agents, perfume, auxiliaries as well as carrier
or combination of any of these besides the HPf and the
encapsulating nano-liposome. This product is referred to as
Lipo-HSP90a.
[0068] The cosmetic compositions of this invention for improving
skin conditions may be formulated in a wide variety of forms, for
example, including a solution, a suspension, an emulsion, a paste,
an ointment, a gel, a cream, a lotion, a powder, a soap, a
surfactant-containing cleanser, an oil, a powder foundation, an
emulsion foundation, a wax foundation and a spray.
[0069] The cosmetically acceptable carrier contained in the present
cosmetic composition, may be varied depending on the type of the
formulation. For example, the formulation of ointment, pastes,
creams or gels may comprise animal and vegetable fats, waxes,
paraffin, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silica, talc, zinc oxide or
mixtures of these substances. In the formulation of powder or
spray, it may comprise lactose, talc, silica, aluminum hydroxide,
calcium silicate, polyamide powder and mixtures of these
substances. Spray may additionally comprise the customary
propellants, for example, chlorofluorohydrocarbons, propane/butane
or dimethyl ether.
[0070] The formulation of solution and emulsion may comprise
solvent, solubilizer and emulsifier, for example water, ethanol,
isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propylene glycol, 1,3-butylglycol, oils, in particular
cottonseed oil, groundnut oil, maize germ oil, olive oil, castor
oil and sesame seed oil, glycerol fatty esters, polyethylene glycol
and fatty acid esters of sorbitan or mixtures of these substances.
The formulation of suspension may comprise liquid diluents, for
example water, ethanol or propylene glycol, suspending agents, for
example ethoxylated isosteary alcohols, polyoxyethylene sorbitol
esters and poly oxyethylene sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar and tragacanth
or mixtures of these substances.
[0071] The formulation of soap may comprise alkali metal salts of
fatty acids, salts of fatty acid hemiesters, fatty acid protein
hydrolysates, isothionates, lanolin, fatty alcohol, vegetable oil,
glycerol, sugars or mixtures of these substances.
[0072] In addition, the cosmetic compositions of this invention may
contain auxiliaries as well as carrier. The non-limiting examples
of auxiliaries include preservatives, antioxidants, stabilizers,
solubilizers, vitamins, colorants, odor improvers or mixtures of
these substances.
[0073] According to the conventional techniques known to those
skilled in the art, the pharmaceutical compositions of this
invention can be formulated with pharmaceutical acceptable carrier
and/or vehicle as described above, finally providing several forms
including a unit dosage form. Most preferably, the pharmaceutical
composition is a solution comprising nano-liposomes.
[0074] The pharmaceutical compositions comprise a pharmaceutically
acceptable carrier. The acceptable carriers include carbohydrates
(e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol,
starch, cellulose), gum acacia, calcium phosphate, alginate,
gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, water, salt solutions, alcohols, gum arabic,
syrup, vegetable oils (e.g., corn oil, cotton-seed oil, peanut oil,
olive oil, coconut oil), polyethylene glycols, methyl cellulose,
methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium
stearate and mineral oil, but not limited to. The pharmaceutical
compositions of this invention, further may contain wetting agent,
sweetening agent, emulsifier, buffer, suspending agent,
preservatives, flavors, perfumes, lubricant, stabilizer, or
mixtures of these substances. Details of suitable pharmaceutically
acceptable carriers and formulations can be found in Remington's
Pharmaceutical Sciences (19th ed., 1995), which is incorporated
herein by reference.
[0075] The pharmaceutical composition of this invention is
developed for topical administration onto skin. The correct dosage
of the pharmaceutical compositions of this invention will be varied
according to the particular formulation, the mode of application,
age, body weight and sex of the patient, diet, time of
administration, condition of the patient, drug combinations,
reaction sensitivities and severity of the disease. It is
understood that the ordinary skilled physician will readily be able
to determine and prescribe a correct dosage of this pharmaceutical
compositions. According to a preferred embodiment of this
invention, the suitable dosage unit is to administer once a day
with 10 pg HPf/cm2 of the affected area .about.1 mg/cm2, 1
ng/cm2-10 .mu.g/cm2, most preferably 10 ng/cn 2.about.1
.mu.g/cm2.
EXAMPLES
[0076] The following specific examples are intended for
illustrating the invention and should not be construed as limiting
the scope of the invention as defined by appended claims.
Example 1. Obtaining the Fragment of Hsp90a (Hpf)
[0077] 1-1) Amplification of the HSP90a fragment (HPf1 cDNA
[0078] The 115 amino acids polypeptide (SEQ ID. NO. 1) used in the
present invention is a fragment of HSP90a (UniProt id: P07900), the
sequence spanning from amino acid (aa) no. 236 to aa no. 350 of the
endogenous protein. This fragment is referred to as a fragment HPf.
In order to produce the HPf in a large scale, the HPf gene was
cloned and expressed in E. coli.
[0079] More specifically, to clone the gene from the human cDNA
library, HEK (Human Embryonic kidney) 293 cell line (CRL-1537,
ATCC, USA) was incubated in 6 well plates for 3 days. After the
removal of the culture media TRizol solution (Invitrogen, USA) 1 ml
was added to dissolve the cells, which was then mixed with 200
.mu.l chloroform by strong vortexing for 10 seconds. The mixture
was centrifuged at 12,000.times.g (Centrifuge 5418, Eppendorf, USA)
for 15 minutes. After the supernatant was collected and transferred
to a new E-tube 0.5 ml isopropyl was added and centrifuged at
12,000.times.g for 10 minutes to precipitate the total RNA. The
total RNA was washed with 70% ethanol once then dissolved in water
free of RNAse and DNAse. Such purified RNA was used to construct
the cDNA library. The cDNA was synthesized using Omniscript Reverse
Transcription kit (Qiagen, U.S.A.) following the instruction
provided in the manufacturer's manual. First, the total RNA 1
.mu.g, IX RT buffer, dNTP mix, oligo-dT primers, RNAse inhibitors
and Omniscript Reverse Transcriptase were mixed, then DNase, RNase
free water was added to adjust the volume to 20 .mu.l, which then
was incubated at 37.degree. C. for 60 minutes to obtain the cDNA
library. Using the cDNA library as the template, genes to be cloned
were prepared by amplifying by PCR. The PCR mixture contains IX PCR
buffer, 6.4 .mu.l 2.5 mM dNTP mix, template (cDNA prepared above),
0.8 .mu.l 100 pmole primer stock, (SEQ ID. NO. 4 and 5) and 0.4
.mu.l proofreading Taq polymerase (TAKARA, Japan) in total volume
of 100 .mu.l. The PCR was performed at 95.degree. C., 30 seconds
for denaturing, 60.degree., 30 seconds for annealing, 72.degree.,
45 seconds for amplification, repeating 35 cycles to amplify the
HPf gene. Subsequently the product was analyzed using agarose gel
electrophorosis to verify the amplification of HPf gene. The HPf
nucleotide sequence is encoded by the SEQ ID. NO. 3, and the amino
acid by sequence at SEQ ID. NO. 1.
[0080] 1-2) Preparation of the Recombinant HPf Protein
[0081] The HPf cDNA obtained from the Example procedure 1-1 above
(cDNA of SEQ ID. NO. 3) prepared through amplification with two
primers (SEQ ID. NO. 4 and SEQ ID. NO. 5) was cloned into pNKmut
plasmid (Korean Patent 10-0985746) using restriction enzymes NdeI
and KpnI (FIG. 2-1A).
[0082] To increase the stability and production of HPf, fusion
proteins of HPf were expressed using TRX (Thioredoxin A, pET-32a,
Novagen, USA), MBP (maltose binding protein, GeneScript, USA), or
HGH (human growth hormone, DNA-sequence ID: NM_000515.3) as a
fusion partner fused in front of HPf.
[0083] To facilitate purification of HPf from the fusion protein
with TRX, a cleavage site for either hydroxylamine(Asn/Gly; N/G) or
TEV (Tabacco Etch Virus) was inserted in between TRX and HPf in the
fusion construct.
[0084] To prepare a fusion protein TRX(NGc)-HPf that is a chimeric
construct of HPf coupled to a fusion partner TRX with an internal
hydroxylamine cleavage site, TRX DNA portion TRX(NGc) (SEQ ID. NO.
18) was obtained by performing PCR using primers (SEQ ID. NO. 8 and
SEQ ID. NO. 9) and pET-32a (0.1 .mu.g) as the template following
Example 1-1 above. Similarly HPf DNA portion was obtained by
performing PCR using primers (SEQ ID. NO. 5 and SEQ ID. NO. 11)
following the procedure as in Example 1-1. To combine TRX(NGc) and
HPf DNA's, primers (SEQ ID. NO. 5 and SEQ ID. NO. 8) were adopted
to perform PCR using the 1:1 mixture of TRX(NGc) and HPf as the
template. The subsequent PCR product was subcloned into Expression
Vector pNKmut (Korean Patent 10-0985746) using DNA restriction
enzymes NdeI and KpnI (FIG. 2-1B).
[0085] Likewise, to prepare a fusion protein TRX(TEVc)-HPf that is
a chimeric construct of HPf coupled to a fusion partner TRX with an
internal TEV cleavage site, TRX DNA portion TRX(TEVc) (SEQ ID. NO.
19) was obtained by performing PCR using primers (SEQ ID. NO. 8 and
SEQ ID. NO. 10) and pET-32a (0.1 .mu.g) as the template following
Example 1-1 above. Similarly HPf DNA portion was obtained by
performing PCR using primers (SEQ ID. NO. 5 and SEQ ID. NO. 12)
following the same procedure as in Example 1-1. BamHI restriction
site was also created between TRX and HPf for later ease of cloning
manipulations. To combine TRX(TEVc) and HPf DNA's, primers (SEQ ID.
NO. 5 and SEQ ID. NO. 8) were used to perform PCR using the 1:1
mixture of TRX(TEVc) and HPf as the template. The subsequent PCR
product was subcloned into Expression Vector pNKmut (Korean Patent
10-0985746) using DNA restriction enzymes NdeI and KpnI (FIG.
2-1C).
[0086] TRX(NGc)-HPf or TRX(TEVc)-HPf fusion protein thus produced
in a transformed E. coli cells showed a much greater level of
expression compared to HPf produced without a TRX fusion partner
(FIGS. 3-1A and 3-1B).
[0087] HPf protein's c-terminal deletion mutant--HPf.DELTA.C1 and
HPf.DELTA.C2 is constructed. HPf.DELTA.C1 is a fragment of HSP90a
(part of HSP90a) consisting of 101 amino acids in total comprising
from Glu236 to Glu336 of HSP90a protein (UniProt ID: P0790), which
was eliminated 14 amino acids from HPf in the carboxyl-terminal
(SEQ ID. NO. 20). Also, HPf.DELTA.C2 is a fragment of HSP90a
composed of 87 amino acids in total comprising from Glu236 to
Asp322 (of HSP90a), which has 28 carboxyl-terminal amino acids less
of HPf, resulting in the smallest protein of the present invention
(SEQ ID. NO. 21) (FIG. 1).
[0088] In order to express the HPf.DELTA.C1 recombinant protein,
the HPf cDNA, acquired from the Example procedure 1-1, was used as
the template and Seq. no. 4 and 6 as primers, via a PCR method
described in Example 1-1. The HPf.DELTA.C1 gene with the sequence
identical to seq. no. 22 was thus obtained and was further cloned
into protein expression vector pNKmut (Korean Patent 10-0985746)
using DNA restriction enzymes NdeI and KpnI. (FIG. 2-2A).
[0089] To clone TRX(TEVc)-Hpf.DELTA.C1 fusion protein composed of
Thioredoxin A coupled with TEV protease recognition site,
HPf.DELTA.C1 gene was obtained by running a PCR using HPf cDNA as a
template and seq.no 6 and 12 as primers by following the same PCR
method described in Example 1-1 above. TRX(TEVc)-HPf fusion
protein-expression plasmid and HPf.DELTA.C1 gene produced by the
PCR were digested by BamHI and KpnI DNA restriction enzymes, then
HPf.DELTA.C1 was cloned into HPf gene-eliminated plasmid by
substitution, yielding HGH(TEVc)-HPf.DELTA.C1 fusion
protein-expression plasmid. (FIG. 2-2B).
[0090] In order to express HPf.DELTA.C2 recombinant protein, HPf
cDNA acquired from Example 1-1 was used as the template, and
primers for seq. no. 4 and 7 were used to acquire HPf.DELTA.C2 with
seq. no. 23, by following the PCR methods described in Example 1-1
above. The resultant PCR product thus obtained was cloned into the
protein expression vector pNKmut (Korean Patent 10-0985746) using
DNA restriction enzymes NdeI and KpnI. (FIG. 2-2C).
[0091] To clone TRX(TEVc)-HPf.DELTA.C2 fusion protein composed of
Thioredoxin A coupled with TEV protease recognition site,
HPf.DELTA.C2 gene was obtained by running a PCR using HPf cDNA
obtained from Example 1-1 as the template and seq. no 7 and 12 as
primers by following the same PCR methods described in Example 1-1
above. TRX(TEVc)-HPf fusion protein-expression plasmid and
HPf.DELTA.C2 gene produced by the PCR were digested by BamHI and
KpnI DNA restriction enzymes, then HPf.DELTA.C2 was cloned into HPf
gene-eliminated plasmid by substitution, yielding
HGH(TEVc)-HPf.DELTA.C2 fusion protein-expression plasmid. (FIG.
2-2D).
[0092] TRX(TEVc)-HPf.DELTA.C1 and TRX(TEVc)-HPf.DELTA.C2 were
transformed into E. coli strain for a scaled-up fermentation, then
their respective protein expression was determined by using
SDS-PAGE. Unexpectedly, those two smaller-version proteins,
HPf.DELTA.C1 and HPf.DELTA.C2, were expressed as soluble protein
forms in the cytoplasm while all HPf-containing fusion proteins are
expressed as inclusion body forms (FIG. 3-2C).
[0093] The primers used for all PCR procedures are listed in the
Table 1 below.
TABLE-US-00001 TABLE 1 Sequences of the primers for PCR Seq.
Primers Sequence No. HPf-up
5'-GAGACATATGGAAGAAAAGGAAGACAAAGAAGAAGAA-3' 4 HPf-dn
5'-TATAGGTACCTTAATCAAAAGGAGCACGTCGTGGGACA-3' 5 HPf.DELTA.C1-dn
5'-GGGGTACCTCATTCCAACTGTCCTTCAACTGAA-3' 6 HPf.DELTA.C2-dn
5'-GGGGTACCTCAATCTTCCCAGTCATTGGTCAAG-3' 7 TRX-up
5'-TTAATTCATATGAGCGATAAAATTATTCACC-3' 8 TRX-NGc-
5'-ACCGTTTTTGAACAGCAGC-3' 9 dn TRX-TEVc- 5'- 10 dn
CTGGAAGTACAGGTTTTCGGATCCATTACCGTTTTTGAACAGC AGCAG-3' HPf-NGc-up 5'-
11 GCTGCTGTTCAAAAACGGTGAAGAAAAGGAAGACAAAGAAG AAGAA-3' HPf-TEVc- 5'-
12 up CCATCCGAAAACCTGTACTTCCAGGGTGAAGAAAAGGAAGA CAAAGAAGAAGAA-3'
HGH-Nde- 5'-GAGACATATGTTCCCGACCATCCCGCTGTCT-'9 13 up HGH-7His- 5'-
14 dn TTTCGGATCCAGAACCATGATGATGGTGATGATGATGACCGA
AGCCACAGCTGCCCTC-3' HSP90(full)-
5'-GAGACATATGCCTGAGGAAACCCAGACCCAGACCC-3' 15 up HSP90(full)-
5'-TATAGGTACCTTAGTCTACTTCTTCCATGCGTGAT-3' 16 dn HSP90-
5'-ACTGGCGGAAGATAAAGAGAA-3' 17 5p(mid)
[0094] To express HPf and HPf.DELTA.C2 as fusion proteins coupled
to a MBP (maltose binding protein) fusion partner, MBP-TEV fusion
construct was synthesized as referenced in Paul, et al (2007)
(GeneScript. USA). To facilitate the cloning of MBP with other
genes to be expressed, MBP-TEV was modified by introducing DNA
restriction enzyme sites NdeI, KpnI, and BamHI at the beginning, at
the end, and in between MBP and TEV genes, respectively. (FIG.
2-3A).
[0095] The modified MBP-TEV gene was cloned into the
protein-expression vector pNKmut (Korean Patent 10-0985746) plasmid
by using DNA restriction enzymes NdeI and KpnI.
[0096] pNKmut plasmid containing MBP-TEV fusion construct was
recovered and digested by DNA restriction enzymes BamHI and KpnI to
remove the internal TEV gene. On the other hand, using SEQ ID. NO.
5 and 12 as primers, a HPf gene was obtained by following the PCR
methods described above in Example 1-1. HPf gene thus obtained was
digested by BamHI and KpnI, then inserted into the BamHI-KpnI
digested pNKmut plasmid containing MBP to obtain MBP(TEVc)-HPf
fusion protein-expression plasmid having the sequence identical to
SEQ ID. NO. 24 (FIG. 2-3B).
[0097] By using TEV recognition site, MBP and its coupled HPf
plasmid were transformed into the E. coli fermentation host, RZ4500
(Biotechnology Institute, Korea University, S. Korea), BL21(DE3)
pLyS (Novagen, USA) and RosettaBlue(DE3) (Novagen, USA) cell lines.
MBP(TEVc)-HPf fusion protein expression of the respective
transformant was confirmed using SDS-PAGE. The result showed that
BL21(DE3)pLyS transformant showed the highest level of
MBP(TEVc)-HPf fusion protein expression in E. coli (FIG. 3-3).
[0098] To express HPf fusion construct coupled to HGH (human growth
hormone) gene, HGH gene was obtained through running a PCR using
HGH gene as the template (DNA-sequence ID: NM_000515.3) and SEQ ID.
NO. 13 and 14 as primers by following the same PCR method as
described above in Example 1-1.
[0099] MBP(TEVc)-HPf fusion protein-expression plasmid and HGH gene
obtained through the PCR were digested by DNA restriction enzymes
NdeI and BamHI, then HGH was cloned into the MBP-eliminated plasmid
by substitution, yielding HGH(TEVc)-HPf fusion protein-expression
plasmid with the sequence identical to SEQ ID. NO. 25 (FIG. 2-4).
The cloned HGH(TEVc)-HPf fusion protein-expression plasmid was
transformed into RZ4500 E. coli cell line (Biotechnology Institute,
Korea University, S. Korea) for a scaled-up fermentation. It was
observed that a large quantity of the fusion protein was expressed
(FIG. 3-4A). It was also confirmed that HGH(TEVc)-HPf fusion
protein was expressed as an inclusion body form within E. coli
(FIG. 3-4B).
[0100] Explanation/Description for each line of FIG. 3-4B. 1:RZ4500
strain (negative control group), 2:HGH(TEVc)-HPf-overexpressing E.
coli strain, 3:Homogenized HGH(TEVc)-HPf-overexpressing E. coli by
sonication, 4. Supernatant from centrifugation of
sonication-homogenized E. coli, 5: Supernatant collected by
centrifugation of the inclusion body re-suspended by washing
solution.
[0101] Expression of the full HSP90a protein (732 amino acids) was
attempted in E. coli Partial carboxy-terminal fragment of full
HSP90a gene was obtained by running a PCR using EST (Expressed
Sequence Tag, clone id: IRCMP5012A0834D) clone containing full
HSP90a gene (full coding region, DNA-sequence ID: NM_001017963) as
a template and SEQ ID. NO. 16 and 17 as primers by following the
same methods as described above in Example 1-1 (FIG. 2-5 A). The
partial carboxy-terminal fragment of full HSP90a was subcloned into
the plasmid pNKmut (Korean Patent 10-0985746) protein-expression
vector by using DNA restriction enzymes NdeI and KpnI. To complete
subcloning of the full HSP90a gene, another PCR was run again using
the EST clone as the template and SEQ ID. NO. 15 and 16 as primers
by following the same methods described above in Example 1-1 (FIG.
2-5A). The PCR products thus acquired was introduced into the NdeI
DNA restriction enzyme-digested site of the plasmid containing
c-terminal part of HSP90a, resulting in construction of the full
HSP90a protein expression plasmid encoding the sequence of HSP90a
identical with SEQ ID. NO. 26 (FIG. 2-5B).
[0102] Their sequences were analyzed using DNA sequencing
confirming the 100% identity to the original sequences of TRX, MBP,
hGH, and HPf. The recombinant cDNA constructs were expressed in the
RZ4500 cell line (Biotechnology Institute, Korea University, S.
Korea), BL21(DE3)pLyS (Novagen, USA), and RosettaBlue (DE3)
(Novagen, USA) to obtain the transformants which were then cultured
in 5 ml LB (Luria-Bertani) media at 37.degree. C. for 16 hrs.
[0103] The protein amount of expressed HPf, TRX(NGc)-HPf,
TRX(TEVc)-HPf, MBP(TEVc)-HPf, and hGH(TEVc)-HPf were analyzed by
SDS-PAGE, of which results reconfirmed the excellent expression of
TRX(TEVc)-HPf gene in BL21 (DE3)pLyS. Therefore, this transformant
is demonstrated to produce the recombinant TRX(TEVc)-HPf fusion
protein in a large scale (FIGS. 3-1, 3-2, 3-3, and 3-4).
Example 2. Confirmation of the Expression of Recombinant Hpf
Protein by Immunoblot
[0104] In order to further confirm whether the expressed
recombinant protein described in the Example 1 is HPf, and
originated from HSP90a, an immunoblot was performed (FIG. 4).
[0105] The transformants expressing the recombinant HPf,
TRX(TEVc)-HPf, HGH (TEVc)-HPf, and full HSP90a genes were cultured
in 5 ml LB media containing ampicillin by shaking at 37.degree. C.
for 16 hours. The culture was centrifuged and the sample was
analyzed with SDS-PAGE. The resulting electrophoresis gel was
analyzed, first, by transferring proteins on the gel to PVDF filter
(Millipore, USA) at 12V for 150 minutes by electrophoresis. Once
the transfer is completed, the filter was then immersed in the
blocking buffer (10% fat free milk and 0.02% Tween 20 and Tris
saline buffer) for 1 hour to inhibit any nonspecific binding. Then
the PVDF filter was immersed in the solution containing the HPf
specific antibody (Rabbit anti-HSP90 antibody, CalbioChem, USA) at
room temperature for 90 minutes. The nonspecific binding was
eliminated by washing the filter for 10 min for three times in
washing buffer (0.02% Tween 20 and Tris saline buffer).
Subsequently the secondary antibody, goat anti-immunoglobulin
antibody (HRP-linked, KOMA, Korea), was added to the reaction
solution and incubated for 1 hour before the filter was washed with
the washing buffer three times. By final staining with
Chemiluminescence, LAS-4000 (Fuji, Japan) immunoblot results
reconfirmed that the expressed protein was HPf. As seen in FIG. 4A,
the recombinant HPf alone and recombinant TRX(TEVc)-HPf proteins
were recognized by the antibody confirming their identity. The
HGH(TEVc)-HPf and Full HSP90a recombinant protein was also
recognized by the specific antibody, anti-HSP90a (FIG. 4B).
Example 3. Large Scale Preparation of HPf
[0106] The host cell line RX4500 transformed with the vector
construct containing the HPf gene TRX(TEVc)-HPf as described in the
Example 1 above, was used to determine the optimum conditions for
the maximum expression of the recombinant protein. Specifically,
the above RZ4500 transformant was cultured in an 1 liter flask,
initially in 7 liter fermentator (FMT-07/C-B, Fermentec, Korea),
which was gradually increased to final 50 L fermentator (FMT-50,
Fermentec, Korea). The culture mixture of the 50 liter fermentator
contains compositions described in the Table 2 below.
TABLE-US-00002 TABLE 2 Fermentation mixture for preparing the
recombinant TRX(TEVc)-HPf protein Compounds % (W/V) NaHPO.sub.4 0.7
KH.sub.2PO.sub.4 0.3 NH.sub.4Cl 0.1 NaCl 0.05 NaNO.sub.3 0.1 Yeast
extract 4 Glycerol 2 Water to 100 pH 7.2
[0107] The seed culture prepared with 1 ml RZ4500 transformed with
TRX(TEVc)-HPf (glycerol stock) was added to 500 ml LB media (pH
7.4) in a 2 liter flask by shaking for 6 hours 37.degree. C. until
the OD600 reached 0.5.about.0.6. For 50 liter fermentation, a
subculture was prepared by mixing the seed culture and culture
media in a ratio of 1:100.
[0108] The concentration of dissolved oxygen in the 50 liter
culture was decreased gradually as the culture time increased.
After 15 hours, the dissolved oxygen concentration remained in the
culture was 10% of the concentration measured immediately after
adding seed culture (FIG. 5). At that time 100-200 ml autoclaved
100% glycerol was added to the culture in order to supplement the
carbon source for the host cell.
[0109] During the 15 hours of fermentation, a portion of culture
was sampled every hour to analyze the pH, dissolved oxygen, and the
O.D. values to determine the growth curve of the host cell (FIG.
5). When the O.D. reached 35-40, the fermentation was terminated.
Also a portion of the culture was analyzed by SDS-PAGE and staining
with Coomassie Brilliant Blue.
[0110] Subsequently, the expression level was quantitatively
determined by measuring the protein concentration of the culture
vs. the BSA (Bovine Serum Albumin, Sigma, USA) with predetermined
concentrations using densitometer (Total Lab Quant, Totallab, USA).
The concentration of the recombinant protein was 1 g/L.
Example 4. Purification and Optimization of the HPf Protein
[0111] The recombinant cells harvested from the large quantity
fermentation was homogenized using homogenizer and washed in 0.5%
Triton X-100 using ultracentrifuger (Hanil, Korea). The inclusion
body was harvested by collecting the precipitate after removing the
supernatant. Then it was dissolved in 25 mM NaOH, renatured with 1%
acetic acid, and centrifuged. Only the supernatant was collected to
remove impurities. Throughout the purification steps a portion of
solutions was removed for analyzing by SDS-PAGE and Coomassie
Brilliant Blue.
[0112] As seen in FIG. 6, HPf was expressed as TRX(TEVc)-HPf in the
inclusion body rather than in the cytosol (lanes 4 and 5), of the
host cell. Its protein structure remained intact during the
denaturation with NaOH and renaturation with acetic acid (lane 7).
Subsequently, TEV protease was added (TRX(TEVc)-HPf: protease=10:1)
and incubated at 4.degree. C. for 24 hours to isolate the HPf from
the TRX(TEVc)-HPf chimeric protein. The cleavage of the chimera by
TEV protease was confirmed by SDS-PAGE as shown in lanes 8-11. The
HPf protein was isolated by gel filtration chromatography
(GFC).
[0113] Lanes of electrophoresis results of FIG. 6 indicate the
proteins: 1. Marker proteins; 2. Competent cell (negative control);
3. Whole cell lysate; 4. Supernatant fraction after the
homogenization (cytosol fraction); 5. Pellet obtained after
homogenization (inclusion body fraction); 6. Supernatant after
washing the pellet; 7. Dissolved inclusion body; 8-11 Solubilized
inclusion body treated with TEV protease. The purity of purified
HPf was >95% as determined by HPLC and SDS-PAGE. The yield after
the purification was determined to be 0.1-0.2 g/liter (FIG. 7).
Example 5. Analysis of HPf by MALDI-TOF
[0114] In order to ensure that the HPf protein from the final
purification step was originated from HPS90a, the MALDI-TOF
analysis (Voyager-DE STR, Applied BioSystems, USA) was performed.
After the electrophoresis of purified HPf (FIG. 7b), the bend
corresponding to HPf was cut out from the gel with a sharp razor.
Then the gel was immersed in 0.1 M (NH.sub.4)HCO.sub.3 solution for
1 hr. After the supernatant was removed, the gel was transferred to
50% acetonitrile in 0.1 M (NH.sub.4)HCO.sub.3 solution for 1 hr,
then in 100% acetonitrile for 15 minutes. Then the in-gel trypsin
digest was performed by mixing the gel with
protein-sequencing-grade trypsin (Promega, USA) in 25 mM
(NH.sub.4)HCO.sub.3 for 16 hours at 37.degree. C. Subsequently 5%
TFA solution containing 60% acetonitrile was added to terminate the
reaction and the mixture was centrifuged. The supernatant was
retrieved to determine the molecular weight by the MALDI-TOF
analysis. According to the analysis using the protein mass
database, the purified protein HPf is a fragment of HSP90a (FIG.
8).
Example 6-1. Analysis of the HPf Particle Size
[0115] In order to prepare the nano-liposome encapsulated HPf for
the pharmaceutical/cosmetic formulation the HPf particle size was
measured using Electrophoretic Light Scattering Spectrophotometer,
ELS-8000.
[0116] HPf from the final purification step was diluted to 1 mg/ml
(or higher concentration) in phosphate buffered saline; pH 7.2, the
light scattered intensity, the weight and number of particles were
determined using ELS 8000. As shown in the FIGS. 9A-9C, the
diameter of the particle was measured to be approximately 10-14 nm.
The diameter of the three dimensional structure of HPf monomer was
approximately 4.4 nm based on the analysis using the software UCSF
Chemera program.
[0117] Since the size of monomer and the HPf in solution could be
different due to its tendency to oligomerize in solution, its size
in solution was measured by gel filtration chromatography. The
results demonstrating the peak of HPf immediately following the
Blue Dextran (200 kDa, Sigma-Aldrich, USA) indicated that HPf does
exist in solution as an oligomeric form (FIG. 9-ID).
Example 6-2. HPf Protein-Size Analysis Via Electron Microscopy
[0118] By using a transmission electron microscope (EF-TEM; Energy
Filtering-Transmission Electron Microscope, KB SI, Korea), HPf
protein particle's size and image were analyzed. The first fixation
process was completed by using a 2.5% glutaraldehyde and 4%
paraformaldehyde solution, and it was washed with a phosphate
buffer solution. Then, the second fixation process was done using
with 1% osmium tetroxide, and underwent dehydration steps beginning
with 60% ethanol, onto 70%, 80%, 90%, 95% and 100% in ascending
order. After embedding with epoxy resin, sample sections were
prepared by thin microslicer. Grids were prepared for section
platform, and samples were observed after the electrostaining
steps. (FIG. 9-2).
Example 7. Evaluation of the Safety of HPf Using Skin Cell
Lines
[0119] To determine whether HPf is safe for human application,
human epidermal cell line (HaCaT) (Schoop, Veronika M., Journal of
Investigative Dermatology., 112 (3):343-353, 1999) and dermal cell
line (HEF) (CRL-7039, ATCC, USA) was incubated with HPf. The
concentration of HPf used for testing the toxicity was 10-100 times
higher than the concentration of epidermal growth factor used in
cosmetic products manufactured and marketed by Regeron Inc. (1-10
.mu.g/ml EGF used for the Clairesome-EF product line).
Specifically, in 96 well plate 2.about.10.times.10.sup.3 cells of
each cell line were plated and cultured in DMEM media (Hyclone,
USA) containing 10% FBS (Fetal Bovine Serum Albumin, Hyclone, USA).
HPf was then added to each cell at the concentration of 0, 0.0001,
0.001, 0.01, 0.1, 0.5, 1, 5, 50 and 100 .mu.g/ml, and the plate was
incubated at 37.degree. C. in the CO.sub.2 incubator for 1 week.
The growth rate (%) of cells was determined by mixing 10 .mu.l
culture media with 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide, Sigma-Aldrich, USA) and incubated at
37.degree. C. in CO.sub.2 incubator for 4 hours. After the
insoluble MTT precipitate was dissolved in 10% Triton X-100 and
0.1N HCl the O.D. was measured using spectrophotometer, spectra MAX
190 (Molecular Device, USA) at 595 nm. The cells mixed with 100
.mu.g/ml of HPf maintained the 90% survival rate (FIG. 10A) after
incubation for 24 hours, and 85% after 7 days incubation (FIG.
10B). The results indicated that HPf can be safely used as a
cosmetic or pharmaceutical ingredient.
Example 8. Stability of HPf in Aqueous Solution
[0120] In order to find out how to prevent the physical/chemical
instability and the loss of bioactivity of HPf in aqueous solution,
the stability of HPf was measured when dissolved in pH 7.2
phosphate buffer solution or kept in a gel state. The gel used for
this analysis was prepared by mixing the following compounds. Care
was taken to exclude any potentially interfering factors that
affect the stability of HPf protein.
TABLE-US-00003 TABLE 3 Composition of the el used for evaluation of
the HPf stability Compounds Amount (%) KOH 0.28 carbomer 0.4
Glycerin 10 phenoxyethanol 0.6 HPf 1 mg/ml Water 88.72
[0121] The stability was evaluated as follows; first, HPf was
diluted to 10 .mu.g/ml in phosphate buffer or to 1 mg/ml while
tested in the gel state. The solution or the gel was left at
4.degree. C. or 37.degree. C. for one month. The amount and/or the
denaturation of the protein was analyzed using Coomassie Brilliant
Blue and SDS-PAGE with samples collected on 3, 7, 14, and 30 days
from the first day of the experiment. The protein stability was
measured using a densitometer (TotalLabQuant, Totallab, USA). As
seen in the FIG. 11, the HPf was consistently stable while kept in
phosphate buffer or in the gel state at 4.degree. C. and 37.degree.
C. for one month.
Example 9. Evaluation of the Efficacy of HPf for Treating Atopic
Dermatitis Using Cell Line Models (Anti-Inflammatory Effects of HPf
Through Suppressing the Degranulation)
[0122] Degranulation of mast cells mediated by IgE is one of the
typical symptoms of atopic dermatitis. To evaluate the
anti-inflammatory effect of HPf, its activity of inhibiting the
secretion of beta hexosaminase, a biomarker for degranulation, was
measured using a basophilic cell line RBL-2H3 (CRL-2256, ATCC,
USA). First, 2.5.times.10.sup.5 RBL-2H3 cells were plated in each
well of 48 well plate and incubated at 37.degree. C. in CO.sub.2
incubator for 3 hours. Cells were sensitized by adding IgE to 1.0
.mu.g/ml and incubated for 24 hours. Then the unbound IgE was
removed by washing the cells with HBS (HEPES buffered Saline) 4
times. Cells were then stimulated by treating with 800.about.1000
ng/2,4-dinitrophenyl hapten-human serum albumin (DNP-HSA, Biosearch
Technologies, USA). Then 50 fl of the supernatant was mixed with
200 fl 0.05M citrate buffer (pH 4.5) containing ImM p-nitrophenyl
N-acetyl-beta-glucosamine and left for 1 hour. The reaction was
terminated by adding 500 fl 0.05M sodium carbonate buffer (pHIO).
The O.D was measured at 405 nm using spectrophotometer. The results
demonstrated (FIG. 12) that 100 .mu.g/ml HPf inhibited the
secretion of beta hexosaminase by 60% when the inhibitory effects
were compared with that of the control (treated with PBS). This
indicates that HPf is able to significantly ameliorate symptoms of
atopic dermatitis by suppressing the degranulation through
inhibiting of the beta hexosaminase secretion.
Example 10. Evaluation of the Efficacy of HPf for Treating Atopic
Dermatitis Using Animal Model
[0123] 10-1) Effects on the Wound Healing
[0124] Atopic dermatitis was induced on the skin of NC/Nga mouse by
topical administration of 150 .mu.l of 0.15%
2.4-dinitrofluorobenzene (DNFB) (dissolved in acetone:olive
oil=3:1) once a week for 4 weeks. The wound healing effects of HPf
was determined as follows: first, mice were divided into two
groups; one group received topical administration of 100 .mu.l HPf
three times per week for 4 weeks, whereas the control group did not
receive HPf. (see FIG. 13A for the scheme of the treatment). The
degree of skin damage (wound) was determined by the naked eye (FIG.
13B), while the infiltration of immune cells and their recovery
were measured by dermal tissue staining (FIG. 14). Compositions
either with or without HPf used for the topical administration was
prepared as gel in order to keep the HPf stay on the applied
area.
TABLE-US-00004 TABLE 4 Composition of the gel used for evaluation
of the HPf efficacy on treating atopic dermatitis. Amount (%) HPf
treated Compounds Control group KOH 0.28 0.28 Carbomer 0.4 0.4
Glycerin 10 10 Phenoxyethanol 0.6 0.6 HPf -- 1 mg/ml Water 88.72
88.72
[0125] Throughout the animal test period, no skin disorders induced
by DNFB were developed except the atopic dermatitis. A topical
administration of DNFB on the skinned back of Nc/Nga mouse induced
the separation of stratum corneum and inflammation due to the wound
(FIG. 13B-2). After 4 weeks of treatment, skin of the treated
group, i.e., the group that received DNFB+HPf (FIG. 13B-4),
appeared virtually wound-free with a slight mark of keratosis,
while the control group demonstrated serious wound left with a
visible sign of inflammation and severe keratosis (FIG. 13B-3).
These results indicate the efficacy of HPf for ameliorating the
symptom of atopic dermatitis.
[0126] 10-2) Effects of HPf for Wound Healing and for Suppressing
the Infiltration of Immune Cells into the Skin Area Affected by
Atopic Dermatitis as Shown in Animal Model
[0127] In order to further evaluate the ability of HPf to heal
wounds on Nc/Nga mouse with atopic dermatitis, a peace of skin
tissue was cut out after 4 weeks from the treatment and stained
with H&E (Hematoxylin & eosin) (FIG. 14). Results
demonstrated a marked separation of stratum corneum in the group
received DNFB only (FIG. 14A-2, 14B-2) or the control group (FIG.
14A-3, 14B-3) without treatment with HPf, while the skin damage was
minimal in the group treated with HPf (FIG. 14A-4, 14B-4) (See the
Table 4 for compositions used for the control group). Atopic
dermatitis is known to cause the infiltration of immune cells
around the affected areas since it tends to secrete various
chemoattractive cytokines. According to the H&E staining
experiment, the group received DNFB only and the control group
without HPf shows the infiltration of immune cells (FIGS. 14A-2,
14B-2 and 14A-3, 14B-3), whereas such an infiltration was minimal
in the group treated with HPf (FIG. 14A-4, 14B-4). The results
strongly suggest that HPf is capable of suppressing the
infiltration of excessive immune cells to the affected areas as
well as healing the wound.
Example 11. Effects of HPf on Skin Cell Differentiation
[0128] Effects of HPf on the cell differentiation of keratinocyte
and fibroblast was evaluated using human epidermal cell line HaCaT
(Schoop, Veronika M, Journal of Investigative Dermatology.,
112(3):343-353) and fibroblast cell line CCD-986sk (SCRL-1947,
ATCC, USA). After each cell line was treated with HPf (100
.mu.g/ml) for 24 hours RNA was extracted and qRT-PCR (SYBR-Green)
was performed.
[0129] Specifically, 0.3.times.10.sup.6 cells/ml were plated in 6
well plates, which was incubated in DMEM (Hyclone, USA) containing
10% FBS until cells reached the 70-80% confluency at 37.degree. C.
in C0 2 incubator. The above cells were treated with HPf to achieve
100 .mu.g/ml as the final concentration and incubated 24 hours.
After removing the supernatant, 1 ml TRizol solution (Invitrogen
USA) was added to dissolve the cells. Then 200 fl chloroform was
added followed by vortexing for 10 sec. and centrifuged at
12,000.times.g (Centrifuge 5418, Eppendorf, USA) for 15 minutes.
The supernatant was collected in a new e tube and mixed with 0.5 ml
isopropyl alcohol and recentrifuged for 10 min. to precipitate the
total RNA. The total RNA was washed with 70% ethanol once then
dissolved in water free of RNAse and DNAse. Such purified RNA was
used to construct the cDNA library. The cDNA was synthesized using
Omniscript Reverse Transcription kit (Qiagen, U.S.A.) following the
instruction provided in the manufacturer's manual.
[0130] First, the total RNA 1 .mu.g, IX RT buffer, dNTP mix,
oligo-dT primers, RNAse inhibitors and Omniscript Reverse
Transcriptase were mixed, then water free of DNase and RNase was
added to adjust the volume to 20 .mu.l, which then was incubated at
37.degree. C. for 60 minutes to obtain cDNAs.
[0131] The expression level of marker genes, such as keratin 10
(KRT10), transglutaminase 1 (TGM1) and involucrin (IVL), which
represents the degree of cell differentiation were determined by
RT-PCR (LightCycler 480, Roche, USA). Sequences of the primers for
RT-PCR are shown in the Table 5. Reagents for the RT-PCR were SYBR
green PCR master mix purchased from Applied Biosystem. The PCR was
initiated by denaturation at 95.degree. C., 10 seconds, followed by
annealing at 60.degree. C., 10 seconds and amplification at
72.degree. C., 10 seconds. The cycle was repeated for 45 times.
[0132] The melting curve analysis was performed at the final cycle
of the RT-PCR to confirm the absence of nonspecific bands. The
RT-PCR products were analyzed ddCt algorithm (A-A-Ct) and the
results are demonstrated in FIG. 15. They indicate that when HaCaT
cells were treated with HPF, the expression level of marker genes
for cell differentiation was 20-250 higher than that of the control
cell (FIG. 15A). In the case of CCD986-sk cell, the expression
level of marker genes was 20 times higher than that of the control
cells (FIG. 15B) when treated with HPf.
[0133] These results suggest that HPf plays important role in
controlling skin cell differentiation hence can be effectively used
as an active ingredient in wound healing medicine and/or cosmetic
products.
TABLE-US-00005 TABLE 5 Sequences of the primers for RT-PCR Seq.
Gene Direction Sequences No. KRT10 Sense 5'-GGTGGGAGTTATGGAGGCAG-3'
28 Antisense 5'-CGAACTTTGTCCAAGTAGGAAGC-3' 29 TGM1 Sense
5'-CATCAAGAATGGCCTGGTCT-3' 30 Antisense 5'-CAATCTTGAAGCTGCCATCA-3'
31 IVL Sense 5'-TCCTCCAGTCAATACCCATCAG-3' 32 Antisense
5'-CAGCAGTCATGTGCTTTTCCT-3' 33
Example 12: Effects of HPf on Subcutaneous Fat Cell Differentiation
In Vitro
[0134] Subcutaneous fat cells secrete various factors necessary to
maintain their structure properly and contain cells that are yet to
be differentiated, such as pre-adipocytes and fat stem cell. We
carried out experiments to find out whether HPf might promote or
suppress the fat cell differentiation. After 3T3-L1 cell line
(CL-173, ATCC, USA) was treated with HPf (100 ug/ml) or with PBS
(pH 7.2) for the control, cells were stained with Oil Red 0 stain
in order to determine the effect of HPf on the fat cell
differentiation. As seen in FIG. 16, cells treated with HPf display
40% reduction in the fat cell differentiation when compared to that
of control cells.
[0135] The present preparations and formulations comprising these
formulations may be used in methods and in specialty preparations
for reducing and/or inhibiting the formation of subcutaneous fat
deposits in an animal, such as in a human.
Example 13. Evaluation of Skin Condition Improving Effects of HPf
Using Artificial Skin
[0136] The present inventors have investigated the effects of HPf
on skin conditioning using artificial skin which has a very similar
3D structure to human skin. The 3D artificial skin culture was
carried out using the Neoderm ED product (TEGO Cell Science Inc.,
Korea) by following the protocols provided by the manufacturer.
Neoderm ED product has epidermal and dermal tissue structure that
is similar to human, hence has been frequently utilized for
developing novel pharmaceuticals for skin as well as cosmetic
products.
[0137] After collagen matrix and fibroblast cells were grown with
media on the surface of cell culture vessels, keratinocytes were
plated on the surface and cultured for 4 days to obtain monolayer
cell. The monolayer of dermal cell was induced by exposing the
cells to air for 16-20 days. Subsequently the artificial dermal
layer was treated with 100 .mu.g ml HPf or with phosphate buffered
saline (Ph 7.2) for 7 days. Then the epidermis and dermis were
stained with H&E stain. FIG. 17 demonstrates that there are no
changes in the thickness of epidermal layers in the control (FIG.
17 A-CI-C3) and HPf treated groups (FIG. 17 B-HI-H4), while the HPf
treated group displayed 2-fold increase in the thickness of dermal
layers when compared to the control groups. This results clearly
indicate that HPf promote the cell differentiation and growth of
dermal layer, implicating that the expression of major skin tissue
components, such as collagen and elastin may be induced by HPf.
[0138] Therefore, HPf can be used as an active ingredient for
improving wrinkles or elasticity, and for developing skin condition
improving cosmetic products.
Example 14. Effects of HPf on Inhibiting Melanin Biosynthesis
[0139] Inhibitory effects of HPf on the melanin biosynthesis were
examined in order to determine whether HPf might be effective on
skin whitening. After B16F10 cell line (CRL-6475, ATCC, USA) was
treated with HPf (IOO.mu.{circumflex over ( )}.SIGMA..tau.I) or PBS
for 48 hours. The cell survival rate and melanin biosynthesis were
measured by MTT assay as shown in FIG. 18. The addition of 100
.mu.g/ml HPf into the culture reduced the melanin biosynthesis 70%
when compared to the control, suggesting that HPf has strong effect
on skin whitening. In a safety test, 100 .mu.g/ml HPf did not
affect the survival of cells indicating no toxicity of HPf at this
concentration.
Example 15. Manufacture of Lipo-HPf, HPf Encapsulated in
Nano-Liposomes
[0140] The following materials were used for manufacturing
Lipo-HPf; soybean lecithin (Shindongbang Inc., Korea) as the
phospholipid, Metarin P (Degussa Texturant Systems Deutschland GmbH
& Co. KG), Nutripur S (Degussa Texturant Systems Deutschland
GmbH & Co. KG) or Emultop (Degussa Texturant Systems
Deutschland GmbH & Co. KG).
[0141] The heat exchanger of a high-pressure homogenizer (max.
output 5 L/hr, highest pressure 1200 bar, Model HS-1002;
manufactured by Hwasung Machinery Co., Ltd., South Korea) was
placed in ice water such that the temperature of the outlet of the
homogenizer did not exceed 30.degree. C., In the meantime the
inside of the homogenizer was then washed with distilled water so
as to be ready to operate. Then, HPf was dissolved in a buffer
solution (20 mM NaH.sub.2PO.sub.4 pH 6.5-7.5, 1 mM EDTA) at a
concentration of 1 mg/ml, phospholipid was added at a ratio of 10
w/v % and sufficiently hydrated and stirred. The stirred solution
was passed through the homogenizer three times or more at room
temperature and a low pressure of 0 bar. To the solution passed
through the homogenizer, phospholipid was added to a ratio of 14
w/v % and sufficiently hydrated and stirred. The stirred solution
was passed through the homogenizer three times or more at 100 bar.
Then, to this solution phospholipid was added to a ratio of 18 w/v
%, sufficiently hydrated and stirred, and passed through the
homogenizer three times or more at 200 bar. Then, to this solution
phospholipid was added to a ratio of 20 w/v %, sufficiently
hydrated and stirred, and passed through the homogenizer three
times or more at 300 bar. Then to this solution, phospholipid was
added to a ratio of 22 w/v %, sufficiently hydrated and stirred,
and passed through the homogenizer three times or more at 400 bar.
Then, to the solution passed through the homogenizer in the
condition of 400 bar, phospholipid was added to a ratio of 24 w/v
%, sufficiently hydrated and stirred, and passed through the
homogenizer three times or more at 500 bar. Then, to the solution
passed through the homogenizer in the condition of 500 bar,
phospholipid was added to a ratio of 26 w/v %, sufficiently
hydrated and stirred, and passed through the homogenizer three
times or more at 600 bar. Then, to the solution passed through the
homogenizer in the condition of 600 bar, phospholipid was added to
a ratio of 28 w/v %, sufficiently hydrated and stirred, and passed
through the homogenizer three times or more at 700 bar. Then this
solution was passed through the homogenizer three times or more at
800 bar followed by centrifugation at 15,000.times.g for 30
minutes. The supernatant was then passed through gel chromatography
(GE Healthcare, USA) to eliminate HPf which was not encapsulated by
liposome, hence preparing HPf-containing liposome (Lipo-HPf) liquid
formulation.
[0142] For a topical preparation, it is envisioned that the product
will include an effective dose estimate of about 100 ng/ml to about
1 mg/ml of each of the HPf polypeptide, the polypeptide fragments,
or a mixture thereof.
Example 16--Drug Delivery Systems (DDS) of Hsp90a and/or Hsp90a
Fragments
[0143] The present example relates to dissolvable microneedle (DMN)
formulations of the heat shock protein preparations that have
parameter dependent conditional monomeric, dimeric and multimeric
association/dissociation/aggregation profiles. In particular, the
heat shock protein Hsp90a and Hsp90a fragments provided herein are
formulated to provide a product of the Hsp90a and/or Hsp90a
fragments in a multitude of dissolvable needles, and in this
manner, provide maximal delivery of the active ingredient to a skin
surface to which it is applied.
[0144] Facial mask formulations that include the micro-needle DMV
formulations of heat shock protein 90a, Hsp90a fragments, or any
combination of these as described herein, are contemplated as a
particular commercial embodiment.
[0145] The drug delivery systems also include parameter dependent
conditional monomeric, dimeric and multimeric
association/dissociation/aggregation profiles of Hsp90a and Hsp90a
fragments. By way of example, some of these formulations may
include noisome formulations, NLC formulations, exosome-liposome
fusion formulations, SLN formulations, NLC formulations,
nanoemulsion formulations, colloidal dispersion formulations and
sprayable formulations.
[0146] Combination products/formulations catered to mesotherapy
devices (microneedle rollers, microinjectors, ultrasonic
cavitation, electroporation, RF-, HF-, sonic vibration massage
rollers, microdermabrasion, laser treatment) may also be provided
that contain the active ingredient Hsp90a and/or Hsp90a fragment
components described herein, such as the HPf polypeptide,
HPf.DELTA.C1, HPf.DELTA.C2, and other fragments and/or fusion
products thereof, to provide delivery of the desired single agent
or combination agent desired.
[0147] Dissolvable microneedle formulations of the present active
ingredients may be prepared according to those techniques known to
those of skill in the art, such as those techniques described in
Moga et al. (2013) (Advanced Materials, 25 (36): 5060-5066).
[0148] Biodegradable microneedles are typically made by filling a
mold with a matrix containing the drug of interest; generally,
multiple vacuum and centrifugation steps are required to completely
fill the molds, arduous steps that lead to lengthy fabrication
times and pose issues to scale-up manufacturing. A thick substrate,
or backing layer, is attached to the array of microneedles to form
a patch. After preparing microneedle patches, they generally are
administered by placing on a skin surface. Conventionally, the
microneedle patch is applied topically to pierce the skin and
penetrate into the viable epidermis or dermis depending on the
physical dimensions of the needles. Due to skin's elastic
qualities, the entirety of the needle does not enter the skin. The
needles are left in the skin for the duration of the treatment
period, from minutes to hours, and the substrate is then removed,
extracting all parts of the needle that have not yet dissolved
(usually 5-20% of each microneedle).
Example 17--Method Development of Functional Assay or BioAssay for
In Vitro Hsp90a or Hsp90a Fragments
[0149] The present example presents an in vitro functional assay or
bioassay for assessing Hsp90a or Hsp90a fragment activity.
[0150] In some embodiments, the functional assay may include a
reference and/or control substrate comprising the Hsp90a, HPf,
HPf.DELTA.C1, or HPf.DELTA.C2 peptide.
Example 18--Methods of Treatment
[0151] The present example presents methods of using the
formulations of Hsp90a and Hsp90a fragments as part of a
therapeutic treatment, alone or together with other therapeutic
agents or treatment modalities. For example, with different Growth
Factor fusion protein partners (for example, growth factors include
human growth factor (hCG), EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF,
GCSF, TGF, Erythropieitn, TPO, BMP, HGF, GDF, Neurotrophins, MSF,
SGF, GDF, to name a few), may be used together with Hsp90a, HPf,
HPf.DELTA.C1, HPf.DELTA.C2, d/or Hsp90a fragments to provide a
combination ingredient for therapeutic use and patient
management.
[0152] By way of example, a therapeutic treatment incorporating
Hsp90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2, and/or Hsp90a fragments,
in a medicament, alone or prepared as a fusion protein in with a
fusion partner protein that is a growth factor (for example, a
fusion protein of the Hsp90 component together with any one or more
of the growth factors recited above, as appropriate), for treatment
of the following conditions:
1) Chronic Wounds (Diabetic foot ulcer, Bed sore, etc.), for
example, alone or together as a topical formulation; 2) Obesity.
For example, the Hsp90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2, and/or
Hsp90a fragment may be incorporated in a dissolvable microneedle
patch preparation, and this preparation may take the form of a
patch or body wrap that may be provided to a skin area or site on a
patient. In this manner, the Hsp90a active ingredient may be
delivered to the patient to reduce fat deposits on the applied
area.
Example 19--Hsp90a and Tau Degradation-Associated Disease,
Co-Chaperones of Hsp90 and Hsp70
[0153] The present example relates to the use of Hsp90a, HPf,
HPf.DELTA.C1, HPf.DELTA.C2, and/or a Hsp90a fragment as part of a
treatment preparation for inhibiting or treating neurodegenerative
diseases (NDD), particularly those associated with tau degradation.
For example, one such neurodegenerative disease associated with tau
degradation is Alzheimers disease (AD).
[0154] Hsp90a, HPf, HPf.DELTA.C1, HPf.DELTA.C2, and/or fragments
thereof, may inhibit formation of a complex of Hsp90 that protects
against tau degradation. For example, inhibitors of Hsp90 have been
reported to decrease levels of phosphorylated tau (Dickey et al.,
2006). Thus, the present investigators provide that Hsp90 may be
used to inhibit the levels of degraded tau levels in a patient by
protecting hyperphosphorylated tau species from degradation.
[0155] A complex of Hsp90 with the co-chaperone FKBP51 was reported
to protect tau from proteasomal degradation and correlated with the
neurotoxic tau species (Jinwal et al., 2010; Blair et al., 2013).
It has also been reported that FKBP51 expression is increased with
age and in Alzheimers disease (Blair et al., 2013). Speculation was
thus made that Hsp90 interaction with FKBP51 is altered in aging
brains and Alzheimers disease brains, allowing for the preservation
of soluble, but possibly neurotoxic protein species. Another member
of FKBP family, FKBP52, may also be involved in tau-related
neurodegeneration. It has been suggested that FKBP52 is a regulator
of tau association with microtubules, specifically that FKBP52
inhibits tau association with microtubules (Chambraud et al.,
2010). A reduction in tau-mediated neurite outgrowth has been
reported in cells overexpressing FKBP52 (Chambraud et al.,
2010).
[0156] The roles of Hsp70, Hsp90, and the co-chaperone STI1, all of
which affect protein folding, will be employed in combination in
the development of Alzheimer disease and other disease (protein
folding disease) therapeutics. The unique cytokine-like activities
of STI1 will be incorporated in these treatment strategies. Both
the extracellular and intracellular activities of STI seem to
converge to increase cellular resilience (Beraldo et al., 2013).
The role of some co-chaperones of Hsp70 in protein misfolding
associated with disease, such as the co-chaperone CHIP, and in high
molecular weight immunophilins, will be used in the development of
appropriate strategies for treatment of protein-misfolding
diseases, as well as other diseases associated therewith.
[0157] Hsp70 promotes tau stability and associates with
microtubules at high levels of expression (Dou et al., 2003; Jinwal
et al., 2009). STI1 may also be important for protection against
aberrant tau species, as its downregulation in fruit flies has been
reported to worsen tau-induced retinal degeneration (Ambegaokar and
Jackson, 2011). Upregulation of both Hsp70 and Hsp90 increases tau
association with microtubules (Dou et al., 2003). Soluble levels of
tau correlate with those of Hsps and their co-chaperones, while in
tauopathies where total levels of tau increase, Hsp70/90 decrease
(Dou et al., 2003). Overall, tau regulation by the Hsp machinery is
very complex and careful analysis of all possible effects on tau is
needed when considering an anti-Alzheimers disease therapy that
modulates this machinery.
[0158] Hsp70 and Hsp90 both interact with many co-chaperones
containing tetratricopeptide repeat (TPR) domains, which consist of
three or more 34-amino acid residues (Lamb et al., 1995). These
motifs form anti-parallel a-helices (Allan and Ratajczak, 2011)
that bind to the C-terminus of the chaperone and are the main
interaction site for co-chaperones (Smith, 2004), along the EEVD
peptide motif on Hsp70 and Hsp90 (Kajander et al., 2009). Proteins
containing TPR domains typically share no other sequence homology,
but are commonly found to be involved in regulation of cell cycle,
protein trafficking, phosphate turnover, and transcriptional events
(Blatch and Lassle, 1999). TPR domain-containing co-chaperones
regulate the ATP cycle of chaperones and aid in client transport to
binding pockets, where they are folded. Hsp40 may help coordinate
other co-chaperones in binding Hsp70, such as Hsp70-interacting
protein (Hip; Hohfeld et al., 1995) in the early stages of the
chaperone cycle, as well as STI1 and SGT (Allan and Ratajczak,
2011). STI1 is also a co-chaperone for Hsp90, along with p23,
Cdc37, and the immunophilins peptidyl-prolyl cistrans (PPIases)
isomerases FKBP51 and FKBP52, phosphatase PP5 and the cyclophillin
Cyp40 (Allan and Ratajczak, 2011). Some of these co-chaperones
inhibit Hsp90 ATP turnover (Rehn and Buchner, 2015). C-terminal
Hsp70-interacting protein (CHIP) is also a co-chaperone for both
Hsp70 and Hsp90.
[0159] Both upregulation of Hsp70 and inhibition of Hsp90 in
mammals reduce protein aggregation and toxicity. STI should be
further investigated in models of protein aggregation, as STI1-PrPC
interaction results in neuroprotection, attenuates AbO toxicity,
and STI1 is an irreplaceable co-chaperone for the Hsp70/Hsp90
machinery.
Example 20--Hsp90a and Anti-Cancer Formulations
[0160] The present example provides for the use of Hsp90a and
Hsp90a fragments in anti-cancer therapeutics. Hsp90a is expected to
provide selective inhibition/affinity against Hsp70/90
co-chaperones-clients complex, which is enriched in numerous
malignant cell models that are absent in normal cells. By
inhibition of this complex, malignant cells may be inhibited, along
with the cancer linked to the identified malignant cell type.
[0161] The purine derivative PU-H71 possesses unique selectivity
amongst Hsp90 inhibitors, preferentially targeting high
molecular-weight complexes composed of Hsp70/90 and various
co-chaperones and client proteins, which are enriched in numerous
malignant cell models, but absent in nononcogenic tissue (Moulick
et al., 2011; Rodina et al., 2016). The formation of these large
stable chaperone species appears to be cancer specific and
diagnostic proteomic approaches may serve as a method to clinically
screen patients that are most likely to benefit from targeting such
species. Whether large and stable chaperone complexes with
misfolded proteins occur in different neurodegenerative diseases is
under study. Therapeutic approaches targeting chaperones involved
in the Hsp70/90 co-chaperones-clients complex in cancer cells are
proposed in the present example.
[0162] Hsp70 and Hsp90 both interact with many co-chaperones
containing tetratricopeptide repeat (TPR) domains, which consist of
three or more 34-amino acid residues (Lamb et al., 1995). These
motifs form anti-parallel a-helices (Allan and Ratajczak, 2011)
that bind to the C-terminus of the chaperone and are the main
interaction site for co-chaperones (Smith, 2004), along the EEVD
peptide motif on Hsp70 and Hsp90 (Kajander et al., 2009). Proteins
containing TPR domains typically share no other sequence homology,
but are commonly found to be involved in regulation of cell cycle,
protein trafficking, phosphate turnover, and transcriptional events
(Blatch and Lassle, 1999). TPR domain-containing co-chaperones
regulate the ATP cycle of chaperones and aid in client transport to
binding pockets, where they are folded. Hsp40 may help coordinate
other co-chaperones in binding Hsp70, such as Hsp70-interacting
protein (Hip; Hohfeld et al., 1995) in the early stages of the
chaperone cycle, as well as STI1 and SGT (Allan and Ratajczak,
2011). STI1 is also a co-chaperone for Hsp90, along with p23,
Cdc37, and the immunophilins peptidyl-prolyl cistrans (PPIases)
isomerases FKBP51 and FKBP52, phosphatase PP5 and the cyclophillin
Cyp40 (Allan and Ratajczak, 2011). Some of these co-chaperones
inhibit Hsp90 ATP turnover (Rehn and Buchner, 2015). C-terminal
Hsp70-interacting protein (CHIP) is also a co-chaperone for both
Hsp70 and Hsp90.
[0163] This system of co-chaperones implicated in the formation of
the Hsp70/90 co-chaperones-clients complex will be targeted in the
development of strategies for treating specific types of cancers
identified to have a malignant cell population that demonstrates an
inhibition of this complex formation upon exposure to one or a
combination of inhibitory molecules, such as a molecule capable of
diminishing and/or competing with co-chaperones of Hsp90 or Hsp70,
such as CHIP and/or STPI1, necessary for complex formation.
Example 21--Hsp90a and Prion Degradation in Disease
[0164] Hsp90a fragments may inhibit formation of a complex of Hsp90
that protects a-synuclein (in case of PD), huntingtin (in HD),
SOD1/TDP-43, and/or prion degradation (PD). Hsp90 inhibition with
geldanamycin in human cell lines has been shown to counteract
formation and accumulation of a-synuclein oligomers and alleviate
a-synuclein-induced toxicity (Klucken et al., 2004; McLean et al.,
2004; Flower et al., 2005; Luk et al., 2008). Much less is known
about the role of Hsp90 in regulating a-synuclein aggregation. In
vitro experiments have been reported to demonstrate that Hsp90 can
abolish a-synuclein binding to vesicles and promote fibril
formation in an ATP-dependent manner (Falsone et al., 2009). Other
in vitro studies investigating Hsp90 interaction with the A53T
mutant of a-synuclein report that all three Hsp90 domains bind to
and prevent A53T a-synuclein aggregation. However, Hsp90 could not
bind to monomeric or fibrillary synuclein species in this model
(Daturpalli et al., 2013).
[0165] STI is capable of having some of its own chaperone-like
activity, but interaction with Hsp70 or Hsp90 would have a greater
effect on reorganization of toxic a-synuclein species. The
literature reports that increasing Hsp70 levels by activating the
heat shock response or by genetic manipulation would be a suitable
method for reducing a-synuclein toxicity.
[0166] Huntington's Disease (HD): In C. elegans expressing the Q35
aggregate prone-protein, siRNA for Hsp40, Hsp70, Hsp90, or STI1 was
reported to increase the number of Htt aggregates (Brehme et al.,
2014). Chaperones and co-chaperones are presented here to provide
therapeutic targets for HD, used in conjunction with Hsp90, Hsp90a,
or the fragments.
[0167] Amyotrophic lateral sclerosis (ALS) is a group of rare
neurological diseases that mainly involve the nerve cells (neurons)
responsible for controlling voluntary muscle movement. There are a
number of proteins, RNAs and miRNAs dysregulated in ALS. The first
aggregated protein to be identified was Cu/Zn superoxide dismutase
(SOD1; Rosen et al., 1993), then trans-active DNA binding
protein-43 (TDP-43; Arai et al., 2006; Neumann et al., 2006), along
with fused in sarcoma/translocated in liposarcoma (FUS; Kwiatkowski
et al., 2009; Vance et al., 2009), see Blokhuis et al. (2013) for a
more extensive review on toxic protein accumulation in ALS.
[0168] Both Hsp70 and Hsp90 can be co-immunoprecipitated with
TDP-43. Moreover, knockdown of Hsp70 or Hsp90 in human
neuroblastoma cells lead to a significant increase in C-terminal
and phosphorylated TDP-43, which are toxic TDP-43 species known to
aggregate in the cytoplasm (Zhang et al., 2010). Treating HeLa
cells with celastrol, an Hsp90 inhibitor, reduced levels of full
length TDP-43, specifically by impairing Cdc37 (an Hsp90
co-chaperone which aids in client protein docking; Lotz et al.,
(2003), Hsp90 interaction with TDP-43 (Jinwal et al., 2012),
Prion). Specifically, STI1 coordination of Hsp70 and Hsp90 was
responsible for this prion elimination activity, as mutations in
the TPR1 and TPR2 domains of STI1 lead to a drastic increase in
PSI+ propagation. This suggests that STI1 coordination of
Hsp70-Hsp90 as well as Hsp104 activity is required for
disaggregation of yeast prions. Furthermore, STI expression and
activity was also found to reduce toxicity of Rnq1 (a yeast protein
with a glutamine-rich prion domain) prions, RNQ+ (Wolfe et al.,
2013). STI1 recruited RNQ+ prions to foci containing Hsp104,
amyloid like proteins and Hsp40, ultimately buffering toxicity by
these prions. The role of Hsp90 and its co-chaperones in prion
diseases is virtually unknown. STI1 can signal via the prion
protein as discussed above, and prion infection in cells abolishes
STI1 signaling via the prion protein (Roffe et al., 2010).
Interaction of Hsp90 with STI1 also decreases PrPC-dependent STI1
neuroprotection (Maciejewski et al., 2016). Secreted Hsp90 may
interfere with STI interaction with PrPC. STI1 regulates protein
aggregates via its co-chaperone activity (Wolfe et al., 2013). STI1
also has extracellular cytokine-like neurotrophic function. The
effects of STI1 on prion diseases and other neurodegenerative
diseases are complex. The present investigators propose treatment
regimens that target the aggregation of these proteins, and
provides an intervention targeting the chaperone machinery toward
refolding or degradation.
[0169] Having described specific examples of the present invention,
it is understood that variants and modifications thereof falling
within the spirit of the invention may become apparent to those
skilled in the art. The scope of the invention is not intended to
be limited to those embodiments provided in the examples. The
appended claims and their equivalents provide a determination of
the scope of the invention.
BIBLIOGRAPHY
[0170] The following references are specifically incorporated
herein by reference in their entirety. [0171] 1. U.S. Pat. No.
7,951,396 [0172] 2. USPub 20080213346 [0173] 3. USPub 20070081963
[0174] 4. Berke, R., et al. American Family Physician 86 (1]:
35-42. July 2012. [0175] 5. Bolinder, J., et al., J Clin Endocrinol
Metab. September; 57(3):455-61, 1983. [0176] 6. Bos J. D. et al.,
Experimental Dermatology, 2000, 9(3): 165-169. [0177] 7. Capristo C
et al., Allergy, August; 59, Suppl 78:53-60, 2004. [0178] 8. Cheng
C F et al., J Clin Invest, 121(11]:4348-61, 2012. [0179] 9. Dhingra
N et al., J Invest DermatoL, 133(10): 2311-4, 2013 October [0180]
10. Pockley, A. G., The Lancet, 362 (9382): pp. 469-476, 2003.
[0181] 11. Schoop, V. M., J Inv. Derm., 112(3): 343-353, 1999.
[0182] 12. Van Noort, J M, et al., J. Biochem. Cell Biol., 44 (10):
pp. 1670-1679, 2012. [0183] 13. Subcutaneous Tissue. Medical
Subject Headings (MeSH). NLM 5 Jun. 2013. [0184] 14. Paul G.
Blommel, Paul G., Fox, Brian, Protein Expr Purifi., 55(1): pp.
53-68, 2007. [0185] 15. USPub 2011/0318400--Lax [0186] 16. USPub
2007/0081963--Oh [0187] 17. Valastyan and Lindquist (2014), Disease
Models & Mechanisms, 7(1): 9-14. [0188] 18. Moga et al. (2013),
Advanced Materials, 25 (36): 5060-5066.
Sequence CWU 1
1
351115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Glu Lys Glu Asp Lys Glu Glu Glu Lys Glu
Lys Glu Glu Lys Glu1 5 10 15Ser Glu Asp Lys Pro Glu Ile Glu Asp Val
Gly Ser Asp Glu Glu Glu 20 25 30Glu Lys Lys Asp Gly Asp Lys Lys Lys
Lys Lys Lys Ile Lys Glu Lys 35 40 45Tyr Ile Asp Gln Glu Glu Leu Asn
Lys Thr Lys Pro Ile Trp Thr Arg 50 55 60Asn Pro Asp Asp Ile Thr Asn
Glu Glu Tyr Gly Glu Phe Tyr Lys Ser65 70 75 80Leu Thr Asn Asp Trp
Glu Asp His Leu Ala Val Lys His Phe Ser Val 85 90 95Glu Gly Gln Leu
Glu Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala 100 105 110Pro Phe
Asp 1152732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pro
Met Glu Glu Glu Glu1 5 10 15Val Glu Thr Phe Ala Phe Gln Ala Glu Ile
Ala Gln Leu Met Ser Leu 20 25 30Ile Ile Asn Thr Phe Tyr Ser Asn Lys
Glu Ile Phe Leu Arg Glu Leu 35 40 45Ile Ser Asn Ser Ser Asp Ala Leu
Asp Lys Ile Arg Tyr Glu Ser Leu 50 55 60Thr Asp Pro Ser Lys Leu Asp
Ser Gly Lys Glu Leu His Ile Asn Leu65 70 75 80Ile Pro Asn Lys Gln
Asp Arg Thr Leu Thr Ile Val Asp Thr Gly Ile 85 90 95Gly Met Thr Lys
Ala Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys 100 105 110Ser Gly
Thr Lys Ala Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile 115 120
125Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val
130 135 140Ala Glu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu
Gln Tyr145 150 155 160Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr
Val Arg Thr Asp Thr 165 170 175Gly Glu Pro Met Gly Arg Gly Thr Lys
Val Ile Leu His Leu Lys Glu 180 185 190Asp Gln Thr Glu Tyr Leu Glu
Glu Arg Arg Ile Lys Glu Ile Val Lys 195 200 205Lys His Ser Gln Phe
Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys 210 215 220Glu Arg Asp
Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp225 230 235
240Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro
245 250 255Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys
Asp Gly 260 265 270Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr
Ile Asp Gln Glu 275 280 285Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr
Arg Asn Pro Asp Asp Ile 290 295 300Thr Asn Glu Glu Tyr Gly Glu Phe
Tyr Lys Ser Leu Thr Asn Asp Trp305 310 315 320Glu Asp His Leu Ala
Val Lys His Phe Ser Val Glu Gly Gln Leu Glu 325 330 335Phe Arg Ala
Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe 340 345 350Glu
Asn Arg Lys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val 355 360
365Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe
370 375 380Ile Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn Ile
Ser Arg385 390 395 400Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val
Ile Arg Lys Asn Leu 405 410 415Val Lys Lys Cys Leu Glu Leu Phe Thr
Glu Leu Ala Glu Asp Lys Glu 420 425 430Asn Tyr Lys Lys Phe Tyr Glu
Gln Phe Ser Lys Asn Ile Lys Leu Gly 435 440 445Ile His Glu Asp Ser
Gln Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg 450 455 460Tyr Tyr Thr
Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr465 470 475
480Cys Thr Arg Met Lys Glu Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly
485 490 495Glu Thr Lys Asp Gln Val Ala Asn Ser Ala Phe Val Glu Arg
Leu Arg 500 505 510Lys His Gly Leu Glu Val Ile Tyr Met Ile Glu Pro
Ile Asp Glu Tyr 515 520 525Cys Val Gln Gln Leu Lys Glu Phe Glu Gly
Lys Thr Leu Val Ser Val 530 535 540Thr Lys Glu Gly Leu Glu Leu Pro
Glu Asp Glu Glu Glu Lys Lys Lys545 550 555 560Gln Glu Glu Lys Lys
Thr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys 565 570 575Asp Ile Leu
Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu 580 585 590Val
Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala 595 600
605Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr
610 615 620Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro
Asp His625 630 635 640Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu
Ala Asp Lys Asn Asp 645 650 655Lys Ser Val Lys Asp Leu Val Ile Leu
Leu Tyr Glu Thr Ala Leu Leu 660 665 670Ser Ser Gly Phe Ser Leu Glu
Asp Pro Gln Thr His Ala Asn Arg Ile 675 680 685Tyr Arg Met Ile Lys
Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr 690 695 700Ala Asp Asp
Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu705 710 715
720Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp 725
7303351DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 3atggaagaaa aggaagacaa agaagaagaa
aaagaaaaag aagagaaaga gtcggaagac 60aaacctgaaa ttgaagatgt tggttctgat
gaggaagaag aaaagaagga tggtgacaag 120aagaagaaga agaagattaa
ggaaaagtac atcgatcaag aagagctcaa caaaacaaag 180cccatctgga
ccagaaatcc cgacgatatt actaatgagg agtacggaga attctataag
240agcttgacca atgactggga agatcacttg gcagtgaagc atttttcagt
tgaaggacag 300ttggaattca gagcccttct atttgtccca cgacgtgctc
cttttgatta a 351437DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 4gagacatatg gaagaaaagg aagacaaaga agaagaa
37538DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5tataggtacc ttaatcaaaa ggagcacgtc gtgggaca
38633DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6ggggtacctc attccaactg tccttcaact gaa
33733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7ggggtacctc aatcttccca gtcattggtc aag
33831DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ttaattcata tgagcgataa aattattcac c
31919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9accgtttttg aacagcagc 191048DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10ctggaagtac aggttttcgg atccattacc gtttttgaac agcagcag
481146DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11gctgctgttc aaaaacggtg aagaaaagga agacaaagaa
gaagaa 461254DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 12ggatccgaaa acctgtactt ccagggtgaa
gaaaaggaag acaaagaaga agaa 541331DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 13gagacatatg ttcccgacca
tcccgctgtc t 311458DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 14tttcggatcc agaaccatga tgatggtgat
gatgatgacc gaagccacag ctgccctc 581535DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15gagacatatg cctgaggaaa cccagaccca gaccc 351635DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16tataggtacc ttagtctact tcttccatgc gtgat 351721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17actggcggaa gataaagaga a 2118255DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 18atgagcgata
aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60gacggggcga
tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc
120ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc
aaaactgaac 180atcgatcaaa accctggcac tgcgccgaaa tatggcatcc
gtggtatccc gactctgctg 240ctgttcaaaa acggt 25519282DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
19atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg
60gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc
120ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc
aaaactgaac 180atcgatcaaa accctggcac tgcgccgaaa tatggcatcc
gtggtatccc gactctgctg 240ctgttcaaaa acggtaatgg atccgaaaac
ctgtacttcc ag 28220101PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Glu Glu Lys Glu Asp Lys
Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu1 5 10 15Ser Glu Asp Lys Pro
Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu 20 25 30Glu Lys Lys Asp
Gly Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys 35 40 45Tyr Ile Asp
Gln Glu Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg 50 55 60Asn Pro
Asp Asp Ile Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser65 70 75
80Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His Phe Ser Val
85 90 95Glu Gly Gln Leu Glu 1002187PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Glu Glu Lys Glu Asp Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu1
5 10 15Ser Glu Asp Lys Pro Glu Ile Glu Asp Val Gly Ser Asp Glu Glu
Glu 20 25 30Glu Lys Lys Asp Gly Asp Lys Lys Lys Lys Lys Lys Ile Lys
Glu Lys 35 40 45Tyr Ile Asp Gln Glu Glu Leu Asn Lys Thr Lys Pro Ile
Trp Thr Arg 50 55 60Asn Pro Asp Asp Ile Thr Asn Glu Glu Tyr Gly Glu
Phe Tyr Lys Ser65 70 75 80Leu Thr Asn Asp Trp Glu Asp
8522309DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 22atggaagaaa aggaagacaa agaagaagaa
aaagaaaaag aagagaaaga gtcggaagac 60aaacctgaaa ttgaagatgt tggttctgat
gaggaagaag aaaagaagga tggtgacaag 120aagaagaaga agaagattaa
ggaaaagtac atcgatcaag aagagctcaa caaaacaaag 180cccatctgga
ccagaaatcc cgacgatatt actaatgagg agtacggaga attctataag
240agcttgacca atgactggga agatcacttg gcagtgaagc atttttcagt
tgaaggacag 300ttggaatga 30923267DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 23atggaagaaa
aggaagacaa agaagaagaa aaagaaaaag aagagaaaga gtcggaagac 60aaacctgaaa
ttgaagatgt tggttctgat gaggaagaag aaaagaagga tggtgacaag
120aagaagaaga agaagattaa ggaaaagtac atcgatcaag aagagctcaa
caaaacaaag 180cccatctgga ccagaaatcc cgacgatatt actaatgagg
agtacggaga attctataag 240agcttgacca atgactggga agattga
267241527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 24atgaaaatcg aagaaggtaa actggtaatc
tggattaacg gcgataaagg ctataacggt 60ctcgctgaag tcggtaagaa attcgagaaa
gataccggca ttaaagtcac cgttgagcat 120ccggataaac tggaagagaa
attcccgcag gttgcggcaa ctggcgatgg ccctgacatt 180atcttctggg
cacacgaccg ctttggtggc tacgctcaaa gcggcctgtt ggctgaaatc
240accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc
cgtacgttac 300aacggcaagc tgattgctta cccgatcgct gttgaagcgt
taagcctgat ttataacaaa 360gacctgctgc cgaacccacc gaaaacctgg
gaagagatcc cggcgctgga taaagaactg 420aaagcgaaag gtaagagcgc
gctgatgttc aacctgcaag aaccgtactt cacctggccg 480ctgattgctg
ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
540gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt
tgacctgatt 600aaaaacaaac acatgaatgc agacaccgat tacagcatcg
cagaagctgc ctttaataaa 660ggcgaaacag cgatgaccat caacggcccg
tgggcatgga gcaacatcga caccagcaaa 720gtgaattatg gtgtaacggt
actgccgacc ttcaagggtc aaccgtccaa accgttcgtt 780ggcgtgctga
gcgcaggtat taacgccgcc agcccgaaca aagagctggc aaaagagttc
840ctcgaaaatt atctgctgac tgatgatggt ctggaagcgg ttaataaaga
caaaccgctg 900ggtgccgtag cgctgaagag ctacgaagaa gagttggtga
atgatccgcg tattgccgcc 960actatggaaa acgcccagaa aggtgaaatc
atgccgatca tcccgcagat gagcgttttg 1020tggtatgccg tgcgtactgc
ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080gccctgaaag
acgcgcagac tatgattaac ggcgatggtg ctggtctgga agtgctgttt
1140cagggtccgg agctaggatc cgaaaacctg tacttccagg gtgaagaaaa
ggaagacaaa 1200gaagaagaaa aagaaaaaga agagaaagag tcggaagacc
aacaagaaat tgaagatgtt 1260ggttctgatg aggaagaaga aaagaaggat
ggtaacaaga agaagaagaa gattaaggaa 1320aagtacatcg atcaagaaga
gctcaacaaa acaaagccca tctggaccag aaatcccgac 1380gatattacta
atgaggagta cggagaattc tataagagct tgaccaatga ctgggaagat
1440cacttggcag tgaagcattt ttcagttgaa ggacagttgg aattcagagc
ccttctattt 1500gtcccacgac gtgctccttt tgattaa 152725981DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
25atgttcccga ccatcccgct gtctcgtctg tttgacaacg ctatgctccg cgcccatcgt
60ctgcaccagc tggcctttga cacctaccag gagtttgaag aagcctatat cccaaaggaa
120cagaagtatt cattcctgca gaacccccag acctccctct gtttctcaga
gtctattccg 180acaccctcca acagggagga aacacaacag aaatccaacc
tagagctgct ccgcatctcc 240ctgctgctca tccagtcgtg gctggagccc
gtgcagttcc tcaggagtgt cttcgccaac 300agcctggtgt acggcgcctc
tgacagcaac gtctatgacc tcctaaagga cctagaggaa 360ggcatccaaa
cgctgatggg gaggctggaa gatggcagcc cccggactgg gcagatcttc
420aagcagacct acagcaagtt cgacacaaac tcacacaacg atgacgcact
actcaagaac 480tacgggctgc tctactgctt caggaaggac atggacaagg
tcgagacatt cctgcgcatc 540gtgcagtgcc gctctgtgga gggcagctgt
ggcttcggtc atcatcatca ccatcatcat 600ggttctggat ccgaaaacct
gtacttccag ggtgaagaaa aggaagacaa agaagaagaa 660aaagaaaaag
aagagaaaga gtcggaagac aaacctgaaa ttgaagatgt tggttctgat
720gaggaagaag aaaagaagga tggtgacaag aagaagaaga agaagattaa
ggaaaagtac 780atcgatcaag aagagctcaa caaaacaaag cccatctgga
ccagaaatcc cgacgatatt 840actaatgagg agtacggaga attctataag
agcttgacca atgactggga agatcacttg 900gcagtgaagc atttttcagt
tgaaggacag ttggaattca gagcccttct atttgtccca 960cgacgtgctc
cttttgatta a 981262199DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 26atgcctgagg
aaacccagac ccaagaccaa ccgatggagg aggaggaggt tgagacgttc 60gcctttcagg
cagaaattgc ccagttgatg tcattgatca tcaatacttt ctactcgaac
120aaagagatct ttctgagaga gctcatttca aattcatcag atgcattgga
caaaatccgg 180tatgaaagct tgacagatcc cagtaaatta gactctggga
aagagctgca tattaacctt 240ataccgaaca aacaagatcg aactctcact
attgtggata ctggaattgg aatgaccaag 300gctgacttga tcaataacct
tggtactatc gccaagtctg ggaccaaagc gttcatggaa 360gctttgcagg
ctggtgcaga tatctctatg attggccagt tcggtgttgg tttttattct
420gcttatttgg ttgctgagaa agtaactgtg atcaccaaac ataacgatga
tgagcagtac 480gcttgggagt cctcagcagg gggatcattc acagtgagga
cagacacagg tgaacctatg 540ggtcgtggaa caaaagttat cctacacctg
aaagaagacc aaactgagta cttggaggaa 600cgaagaataa aggagattgt
gaagaaacat tctcagttta ttggatatcc cattactctt 660tttgtggaga
aggaacgtga taaagaagta agcgatgatg aggctgaaga aaaggaagac
720aaagaagaag aaaaagaaaa agaagagaaa gagtcggaag acaaacctga
aattgaagat 780gttggttctg atgaggaaga agaaaagaag gatggtgaca
agaagaagaa gaagaagatt 840aaggaaaagt acatcgatca agaagagctc
aacaaaacaa agcccatctg gaccagaaat 900cccgacgata ttactaatga
ggagtacgga gaattctata agagcttgac caatgactgg 960gaagatcact
tggcagtgaa gcatttttca gttgaaggac agttggaatt cagagccctt
1020ctatttgtcc cacgacgtgc tccttttgat ctgtttgaaa acagaaagaa
aaagaacaac 1080atcaaattgt atgtacgcag agttttcatc atggataact
gtgaggagct aatccctgaa 1140tatctgaact tcattagagg ggtggtagac
tcggaggatc tccctctaaa catatcccgt 1200gagatgttgc aacaaagcaa
aattttgaaa gttatcagga agaatttggt caaaaaatgc 1260ttagaactct
ttactgaact ggcggaagat aaagagaact acaagaaatt ctatgagcag
1320ttctctaaaa acataaagct tggaatacac gaagactctc aaaatcggaa
gaagctttca 1380gagctgttaa ggtactacac atctgcctct ggtgatgaga
tggtttctct caaggactac 1440tgcaccagaa tgaaggagaa ccagaaacat
atctattata tcacaggtga gaccaaggac 1500caggtagcta actcagcctt
tgtggaacgt cttcggaaac atggcttaga agtgatctat 1560atgattgagc
ccattgatga gtactgtgtc caacagctga aggaatttga ggggaagact
1620ttagtgtcag tcaccaaaga aggcctggaa cttccagagg atgaagaaga
gaaaaagaag 1680caggaagaga aaaaaacaaa gtttgagaac ctctgcaaaa
tcatgaaaga catattggag
1740aaaaaagttg aaaaggtggt tgtgtcaaac cgattggtga catctccatg
ctgtattgtc 1800acaagcacat atggctggac agcaaacatg gagagaatca
tgaaagctca agccctaaga 1860gacaactcaa caatgggtta catggcagca
aagaaacacc tggagataaa ccctgaccat 1920tccattattg agaccttaag
gcaaaaggca gaggctgata agaacgacaa gtctgtgaag 1980gatctggtca
tcttgcttta tgaaactgcg ctcctgtctt ctggcttcag tctggaagat
2040ccccagacac atgctaacag gatctacagg atgatcaaac ttggtctggg
tattgatgaa 2100gatgacccta ctgctgatga taccagtgct gctgtaactg
aagaaatgcc accccttgaa 2160ggagatgacg acacatcacg catggaagaa
gtagactaa 2199277PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Glu Asn Leu Tyr Phe Gln Gly1
52820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28ggtgggagtt atggaggcag 202923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
29cgaactttgt ccaagtagga agc 233020DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 30catcaagaat ggcctggtct
203120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31caatcttgaa gctgccatca 203222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
32tcctccagtc aatacccatc ag 223321DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 33cagcagtcat gtgcttttcc t
21346PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 34His His His His His His1 5357PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 7xHis tag
35His His His His His His His1 5
* * * * *