U.S. patent application number 14/191479 was filed with the patent office on 2014-06-26 for sp1 polypeptides, modified sp1 polypeptides and uses thereof.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. The applicant listed for this patent is Fulcrum SP Ltd., Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Arie Altman, Or Dgany, Ira Marton, Yehonathan Pouny, Oded Shoseyov, Amnon WOLF.
Application Number | 20140178483 14/191479 |
Document ID | / |
Family ID | 37637586 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178483 |
Kind Code |
A1 |
WOLF; Amnon ; et
al. |
June 26, 2014 |
SP1 POLYPEPTIDES, MODIFIED SP1 POLYPEPTIDES AND USES THEREOF
Abstract
SP1 and modified SP1 variant polypeptides capable of forming
reversible molecular associations with substances,
compositions-of-matter comprising same, and uses thereof are
provided.
Inventors: |
WOLF; Amnon; (Herzlia
Pituach, IL) ; Pouny; Yehonathan; (Givat Shmuel,
IL) ; Marton; Ira; (Rehovot, IL) ; Dgany;
Or; (Ashdod, IL) ; Altman; Arie; (Tel-Aviv,
IL) ; Shoseyov; Oded; (Karmei Yosef, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yissum Research Development Company of the Hebrew University of
Jerusalem
Fulcrum SP Ltd. |
Jerusalem
Herzlia Pituach |
|
IL
IL |
|
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
Fulcrum SP Ltd.
Herzlia Pituach
IL
|
Family ID: |
37637586 |
Appl. No.: |
14/191479 |
Filed: |
February 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11988314 |
Jan 4, 2008 |
|
|
|
PCT/IL2006/000795 |
Jul 9, 2006 |
|
|
|
14191479 |
|
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|
Current U.S.
Class: |
424/491 ;
514/21.2; 530/350 |
Current CPC
Class: |
C07K 14/415 20130101;
Y02A 50/30 20180101; A61K 47/6929 20170801; B82Y 30/00 20130101;
A61K 47/64 20170801; A61K 38/17 20130101; A61P 43/00 20180101; Y02A
50/473 20180101 |
Class at
Publication: |
424/491 ;
530/350; 514/21.2 |
International
Class: |
C07K 14/415 20060101
C07K014/415 |
Claims
1. A composition of matter comprising an SP-1
polypeptide-nanoparticle complex, wherein said SP-1 polypeptide
comprises a boiling- and detergent stable protein having at least
85% sequence identity with SEQ ID NO: 1.
2. A method of coating a surface with a nanoparticle comprising:
(a) contacting the nanoparticle with an SP-1 polypeptide in order
to form a composition of matter comprising an SP-1
polypeptide-nanoparticle complex; and (b) contacting said surface
with the SP-1-nanoparticle complex, thereby coating said surface
wherein said SP-1 polypeptide comprises a boiling- and detergent
stable protein having at least 85% sequence identity with SEQ ID
NO: 1.
3. The method of claim 2, wherein said SP-1 polypeptide is selected
from the group consisting of native SP-1, SP-1 variants comprising
a gold-binding peptide and a SP-1 variants comprising a silicon
binding peptide.
4. The method of claim 2, wherein said SP-1 polypeptide is a mutant
comprising a peptide selected from the group consisting of SEQ ID
NOs. 82-84 and 94-103.
5. The method of claim 2, wherein said nanoparticle is a conducting
molecule or a semiconducting molecule.
6. The composition of matter of claim 1, wherein said SP-1
polypeptide is selected from the group consisting of native SP-1,
SP-1 variants comprising a gold-binding peptide and a SP-1 variants
comprising a silicon binding peptide.
7. The composition of matter of claim 1, wherein said SP-1
polypeptide is a mutant comprising a peptide selected from the
group consisting of SEQ ID NOs. 82-84 and 94-103.
8. The composition of matter of claim 1, wherein said nanoparticle
is a conducting molecule or a semiconducting molecule.
9. A method of enhancing the dispersion of a nanoparticle in a
solvent comprising: (a) contacting the nanoparticle with an SP-1
polypeptide in order to form a composition of matter comprising an
SP-1 polypeptide-nanoparticle complex; and (b) contacting said
SP-1-nanoparticle complex with said solvent, thereby enhancing the
dispersion of said nanoparticles in said solvent, wherein said SP-1
polypeptide comprises a boiling- and detergent stable protein
having at least 85% sequence identity with SEQ ID NO: 1.
10. The method of claim 9, wherein said solvent is an aqueous
solvent.
11. The method of claim 9, wherein said SP-1 polypeptide is a
mutant comprising a peptide selected from the group consisting of
SEQ ID NOs. 82-84 and 94-103.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 11/988,314 filed on Jan. 4, 2008, which is a National
Phase of PCT Patent Application No. PCT/IL2006/000795 having
International Filing Date of Jul. 9, 2006. The contents of the
above applications are all incorporated herein by reference.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 57189SequenceListing.txt, created
on Feb. 23, 2014, comprising 215,552 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention relates to denaturant and protease
stable proteins, modified derivatives thereof, and uses thereof.
More particularly, the present invention relates to the use of
novel denaturant-stable, protease resistant, homo-oligomeric
proteins, also referred to herein as stable proteins (SPs), and
derivatives thereof designed for complexing, release and delivery
of other molecules (ligands) and nanostructures.
[0004] Denaturant-Stable, Protease Resistant Proteins
[0005] A unique family of stress-induced, chaperone-like proteins
having exceptional resistance to harsh conditions has been recently
identified in widely diverse plant species. Exemplified by the SP1
protein of Aspen (SEQ ID NO: 1), this family of proteins is
characterized by boiling-, denaturant- and protease-resistance,
regions of conserved amino acid sequence homology, unique
three-dimensional conformation, oligomer formation and a strong
stabilizing effect on biologically active proteins.
[0006] The exceptional resistance of these stress-induced,
chaperone-like proteins to harsh conditions in combination with
their unique three dimensional structure allows the application of
extreme condition to create stable, but selectively reversible
complexes with the ligand.
[0007] SP1
[0008] SP1, isolated from aspen plants (Populus tremula), responds
to a wide range of environmental stresses, including salinity, cold
and heat stress and accumulates during stress recovery. No
significant sequence similarity has been found with known protein
families and SP1 homologues have been observed in a number of plant
and bacterial species, either as putative proteins from genomic
sequences or ESTs with unknown function.
[0009] Wang et al. (U.S. patent application Ser. No. 10/233,409)
have isolated, cloned and characterized the Aspen SP1 protein (SEQ
ID NO: 1), and uncovered it's chaperone-like activity in
stabilizing other, biologically active proteins against
denaturation. Wang et al (U.S. patent application Ser. No.
10/233,409) further disclosed other boiling and detergent-stable
proteins from other, diverse plant species (Tomato, Pine, Rice,
Corn and Arabidopsis) sharing similar functional characteristics,
specifically, chaperone-like activity and stress-relatedness,
sharing immune-cross reactivity, having at least 65% amino acid
homology to the Aspen SP1, and sharing a conserved region of
sequence homology.
[0010] Wang et al (U.S. patent application Ser. No. 10/233,409)
disclosed SP1 proteins fused to other protein or non-protein
molecules, for enhancement of binding properties of binding
molecules, for stabilization of the fused molecules (such as
enzymes) and for enhancement or alteration of immunological
properties of the fused molecules. SP1 fusion proteins, as taught
by 10/233,409, comprise recombinant SP1 molecules having additional
polypeptide sequences added by genetic engineering techniques, and
SP1 molecules having additional non-protein moieties added by
chemical means, such as cross linking. Wang et al have further
disclosed the therapeutic use of SP1 proteins for strengthening
skin, hair, nails, etc. However, U.S. patent application Ser. No.
10/233,409 do not teach, nor imply, the use of native SP1, or SP1
variants as carriers for and means of controlled release of, agents
(therapeutic, cosmetic, diagnostic, conductive, etc) reversibly
complexed therewith.
[0011] Drug Carriers:
[0012] Many drugs employed to treat diseases are either
insufficiently soluble in aqueous solutions or have adverse side
effects in therapeutic concentrations. Thus, many medical
applications suffer from a lack of suitable methods for efficiently
delivery of effective concentrations of drugs to a target cell or
tissue in an organism (e.g., mammal) in need of treatment.
[0013] Some considerations for efficacious use of drugs
include:
[0014] Poor solubility, causing difficulty in achieving a
convenient pharmaceutical format, as hydrophobic drugs may
precipitate in aqueous media. However, the use of excipients for
solubilization such as Cremphor (the solubilizer for paclitaxel in
Taxol) is also associated with toxicity.
[0015] Lack of selectivity for target tissues, leading to toxicity
to normal tissues, severely restricting the amount of drug that can
be administered, as in the case of the cardiac toxicity of
doxorubicin. Low concentrations of drugs in target tissues further
results in suboptimal therapeutic effects.
[0016] Unfavorable pharmacokinetics, such as rapid renal clearance,
rapid breakdown of the drug in vivo, or loss of activity at
physiological conditions (e.g. loss of activity of camptothecins at
physiological pH), can also lead to heightened dosing or a frequent
administration regimen.
[0017] Development of drug resistance in target tissue, such as
tumors, by induction of cellular transporters, detoxification
pathways, or inhibition of apoptosis transduction pathways.
[0018] Tissue damage on extravasation of cytotoxic drugs, leading
to tissue damage (i.e. necrosis caused by free paclitaxel).
[0019] A number of approaches have resolved some of these issues in
specific cases, but there is yet no general solution to the
problems of drug delivery. Some examples of existing approaches for
solving these problems include (1) solublization of hydrophobic
drugs in micelles formed from surfactants in aqueous media
(Wiedmann and Kamel, J. Pharm. Sci. 2002, 91, 1743; MacGregor, et
al., Adv. Drug Deliv. Rev. 1997, 25, 33), (2) encapsulation of
drugs in polymeric matrices in the nanometer to micrometer size
range which may be biodegradable and may contain bioadhesive
functional groups or ligands (WO 02/15877, WO 02/49676), (3)
encapsulation of hydrophilic drugs in liposomes (Anderson, et al.,
Pharm. Res. 2001, 18, 316; WO 99/33940), which may also display
bioadhesive functional groups or ligands, (4) conjugation of drugs
to molecules that are substrates for active transport systems
(Kramer, et al., J. Biol. Chem. 1994, 269, 10621; WO 01/09163; US
2002/0098999; US 20060074225), (5) targeting using physiologically
selective (pH, enzymatic, etc.) release of active drug components
(i.e. prodrugs), (6) association of the drug with hydrogels and (7)
chemical derivatization of protein drugs with hydrophilic polymers
to protect them from degradation, immune recognition, or renal
excretion (Belcheva, et al., Bioconjugate Chem. 1999, 10, 932;
Zalipsky, Bioconjugate Chem. 1995, 6, 150; U.S. Pat. No. 4,002,531;
U.S. Pat. No. 4,179,337). None of these approaches, however, offers
a general solution for all cases of drug delivery problems. Control
of particle size in micellar, liposomal, and polymeric
nanoparticulate systems remains a serious problem. The inability of
currently available drug delivery systems to incorporate all of the
functions required for delivery into a single system is another
problem with for example, micelles, nanoparticulate systems and
targeted systems. Yet further, the release rate and storage life,
especially of micelles and liposomes, is difficult to control and
unpredictable, and amphiphylic components can produce toxic
effects.
[0020] Other systems employed for drug delivery to a cell or tissue
of an organism have similar drawbacks. Thus, there is a need for a
method to deliver drugs that minimize or overcome the
above-referenced problems.
[0021] The invention includes methods for the use of SP1 and SP1
variants for forming molecular complexes with other substances such
as small molecules, peptides, nucleic acid fragments, inorganic
nanostructures and other molecules (ligands). In addition the
invention includes methods for the use of SP1 and SP1 variants for
molecular complexing of drugs and delivery as well as control
release of complexed ligands. There is thus a widely recognized
need for, and it would be highly advantageous to have, SP1 and SP1
variants capable of forming molecular complexes devoid of the above
limitation.
SUMMARY OF THE INVENTION
[0022] According to one aspect of the present invention there is
provided an isolated polypeptide comprising an amino acid sequence
of an SP1 polypeptide, said amino acid sequence being modified to
be in a reversible molecular association with a substance.
[0023] According to yet another aspect of the present invention,
there is provided an isolated polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO:2-30.
[0024] According to still another aspect of the present invention,
there is provided an isolated polynucleotide comprising a nucleic
acid sequence encoding a polypeptide having an amino acid sequence
as set forth in SEQ ID NO: 2-30, or an isolated polynucleotide
encoding an isolated polypeptide comprising an amino acid sequence
of an SP1 polypeptide, said amino acid sequence being modified to
be in a reversible molecular association with a substance.
[0025] According to another aspect of the present invention, there
is provided a composition of matter comprising a plurality of
self-assembled modified SP1 monomers.
[0026] According to yet another aspect of the present invention,
there is provided an isolated composition-of-matter comprising a
therapeutic, diagnostic or cosmetic agent being in molecular
association with a modified SP1 polypeptide.
[0027] According to further features in the preferred embodiments
of the invention described below, the SP1 molecule is
translationally fused to the agent.
[0028] According to still another aspect of the present invention,
there is provided an isolated composition-of-matter comprising an
SP1 polypeptide in reversible molecular association with a
therapeutic, diagnostic or cosmetic agent.
[0029] According to further features in the preferred embodiments
of the invention described below, the SP1 molecule is not
translationally fused to the agent.
[0030] According to yet another aspect of the present invention,
there is provided an isolated composition-of-matter comprising a
conductive or semi-conductive substance being in molecular
association with a modified SP1 polypeptide.
[0031] According to yet another aspect of the present invention,
there is provided an isolated composition-of-matter comprising an
SP1 polypeptide in reversible molecular association with a
conductive or semi-conductive substance.
[0032] According to yet another aspect of the present invention,
there is provided a method of delivering a therapeutic, diagnostic
or cosmetic agent to a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of the composition of matter of comprising a therapeutic,
diagnostic or cosmetic agent being in molecular association with a
SP1 polypeptide to the subject, thereby delivering said
therapeutic, diagnostic or cosmetic agent to said subject.
[0033] According to further features in the preferred embodiments
of the invention described below the SP1 polypeptide can be a
modified SP1 polypeptide.
[0034] According to yet further features in the preferred
embodiments of the invention described below, the molecular
association is a reversible molecular association, and the method
further comprising providing conditions for reversing said
molecular association.
[0035] According to yet another aspect of the present invention,
there is provided a method of stabilizing a substance, the method
comprising contacting the substance with an SP1 polypeptide
modified to reversibly form a complex with said substance so as to
form a complex, thereby stabilizing the substance.
[0036] According to further features in the preferred embodiments
of the invention described below, the stability comprises a
property selected from the group consisting of temperature
stability, ionic strength stability, protease stability and
catalytic stability.
[0037] According to further features in the preferred embodiments
of the invention described below, the method further comprising the
step of contacting said complex with a solvent, so as to form a
solution.
[0038] According to yet another aspect of the present invention,
there is provided a method of enhancing the solubility of a
substance in a solution. The method is effected by contacting the
substance with an SP1 polypeptide capable of reversibly forming a
complex with said substance so as to form a complex and dissolving
said complex with a solvent so as to form a solution, thereby
enhancing the solubility of the substance in the solution. The
solvent can be an aqueous or organic solvent, and the substance can
be a hydrophobic or hydrophilic substance.
[0039] According to further features in the preferred embodiments
of the invention described below, the SP1 polypeptide is a boiling
and detergent stable protein at least 65% homologous to SEQ ID NO:
1, said boiling and detergent stable protein having a
chaperone-like activity and being capable of forming stable
dimers.
[0040] According to yet further features in the preferred
embodiments of the invention described below the SP1 polypeptide
has at least one conserved amino acid sequence in at least one
region corresponding to amino acids 9-11, 44-47 and/or 65-73, of
SEQ ID NO:1, as determined using a Best Fit algorithm of GCG,
Wisconsin Package Version 9.1, using a plurality of 10.00, a
threshold of 4, average weight of 1.00, average match of 2.91 and
average mismatch of minus 2.00.
[0041] According to still further features in the preferred
embodiments of the invention described below the SP1 polypeptide is
characterized by oligomer formation, and the SP1 oligomer is a heat
stable and protease resistant oligomer.
[0042] According to further features in the preferred embodiments
of the invention described below the SP1 polypeptide is an SP1
polypeptide having a modified amino acid sequence, and the
modification comprises addition of at least one amino acid capable
of forming disulfide bonds.
[0043] According to further features in the preferred embodiments
of the invention described below the modification comprises
addition of at least 2 histidine residues at a position
corresponding to amino acid residue 2 of SEQ ID NO: 1.
[0044] According to still further features in the preferred
embodiments of the invention described below the modification
comprises the addition of at least one amino acid having at least
one thiol group at a position corresponding to amino acid residue
40 of SEQ ID NO:1.
[0045] According to yet further features in the preferred
embodiments of the invention described below, forming the molecular
association or complex with the agent is redox-dependent.
[0046] According to still further features in the preferred
embodiments of the invention described below the modification is an
addition of a cysteine residue at a position corresponding to amino
acid residue 2 or 40 of SEQ ID NO:1.
[0047] According to further features in the preferred embodiments
of the invention described below the substance is a therapeutic
agent, a diagnostic agent or a cosmetic agent. Yet further, the
therapeutic agent, diagnostic agent or cosmetic agent is selected
from the group consisting of a polypeptide agent, a nucleic acid
agent, a lipid agent, a carbohydrate agent, a small molecule and a
combination of same.
[0048] According to further features in the preferred embodiments
of the invention described below, the substance is a conductive or
semiconductive ionic substance. The conductive or semiconductive
ionic substance can be any of metals, semiconductors and
dielectrics.
[0049] According to further features in the preferred embodiments
of the invention described below, the modified amino acid sequence
is as set forth in SEQ ID NO:2-30.
[0050] According to further features in the preferred embodiments
of the invention described below, the said amino acid sequence is
modified to comprise a target recognition sequence. The target
recognition sequence can be a cancer cell surface or cancer cell
vasculature recognition sequence. The target recognition sequence
can be any of the sequences of SEQ ID NOs: 31-62. The target
recognition sequence can be a cancer cell vasculature recognition
sequence is selected from the group consisting of SEQ ID NOs:
63-81. The cancer cell vascular recognition sequence can be a CRGD
(SEQ ID NO: 151) sequence.
[0051] According to further features in the preferred embodiments
of the invention described below, the molecular association is a
covalent association or a non-covalent association.
[0052] According to yet another aspect of the present invention,
there is provided the use of a composition of matter comprising a
therapeutic, diagnostic or cosmetic agent being in molecular
association with a native or a modified SP1 polypeptide for the
manufacture of a medicament for delivering a therapeutic,
diagnostic or cosmetic agent to a subject in need thereof.
[0053] According to still another aspect of the present invention,
there is provided a of a composition of matter comprising a
therapeutic, diagnostic or cosmetic agent being in molecular
association with a native or a modified SP1 polypeptide for the
delivering a therapeutic manufacture of a medicament for delivering
a therapeutic, diagnostic or cosmetic agent to a subject in need
thereof.
[0054] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
isolated SP1 polypeptides and SP1 variant polypeptides capable of
forming molecular associations with substances for use in
therapeutic, diagnostic, cosmetic and nano-technonological
applications, such as drug delivery, solubilization and
stabilization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0056] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0057] In the drawings:
[0058] FIG. 1 is an electron micrograph of a negatively stained 2D
crystal of SP1 oligomer, and a graphic representation of the 2D
crystalline structure;
[0059] FIGS. 2a-2b are electron micrographs of recombinant SP1:
6HSP1 showing binding of NTA-Ni gold nano-particles to the 6H tags
in the central cavity. FIG. 2a is a transmission electron
micrograph (TEM); Note the alternating sequence of
Protein-nanogold-Protein-nanogold . . . ; FIG. 2b is a graphic
representation of the nanogold-6HS-SP1 conjugate;
[0060] FIG. 3 is a SDS-PAGE showing the self-assembly of two SP1
variants into hetero-oligomer SP1 complexes. Monomers of
recombinant HIS-tagged SP1 (6H) and N-terminal deleted SP1
(.DELTA.N) were co-electro-eluted (6H.DELTA.N), and purified on the
Ni-NTA Agarose and subjected to Proteinase K (PK) digestion.
Protein samples were prepared in SDS-sample buffer with excess
ratio of SDS to the sample, either boiled (b) for 5 min, or without
boiling (nb). Lane 1=WT: wild type SP1. Boiled, monomeric, gel
purified forms of 6HSP1 (lane 2) and .DELTA.NSP1 (lane 3) were
visualized by Coomassie blue staining, excised and mixed at 1:1
ratio (v/v) (lanes 4 and 5). The proteins were co-eluted by
electro-elution as described previously (Wang (2002)). The
hetero-oligomeric complex was isolated on Ni-NTA Agarose beads
(lanes 6 and 7). Proteinase K which digests monomeric SP1 but not
SP1 complex, was employed to eliminate the monomeric form of 6HSP1
and .DELTA.NSP1 from the Ni-NTA purified proteins (lanes 8 and 9).
The composition of hetero-olimeric SP1 was determined by SDS-PAGE
and visualized by silver staining;
[0061] FIG. 4 shows the multiple display of the tumor specific
peptides CRGD (SEQ ID NO: 151) and RGDC (SEQ ID NO: 152) on SP1
surface. The peptides were inserted to SP1 N-terminus. Note that in
both the CRGD (lanes 1-3) (SEQ ID NO: 5), and the RGD-C (lanes 5-7)
(SEQ ID NO: 6) variants form high molecular weight complexes (lanes
3 and 7). When the protein is boiled in the sample application
buffer in the presence of reducing agent the complex is dissociated
to lower molecular weight species (monomer, dimer and others). In
the absence of reducing agents (lanes 1 and 5), and boiling under
oxidizing conditions (in the absence of reducing agents) higher
levels of dimers are observed (lane 2). Lane 4=molecular weight
markers;
[0062] FIGS. 5a and 5b are photographs of PAGE analysis showing the
expression, purification and refolding of recombinant variant SP1.
Cys2loop1RGd SP1 variants not forming soluble protein during
expression (found in inclusion bodies, IB) extracted by French
press and sedimented by centrifugation (20 mM. 17,000.times.g). The
supernatant which does not contain soluble SP1 (FIG. 5a, lane 1)
was collected, and the pellet which contain the SP1 IB (FIG. 5a,
lane 2) was washed in dilute urea, and solubilized in 5M urea and
10 mM DTT, centrifuged (30 min. 20000.times.g). The pellet (FIG.
5a, lane 3) was discarded, and the supernatant was collected and
dialyzed against buffer with 2 mM DTT for four days (FIG. 5a, lane
4). The dialyzed variant SP1 was stored in cold 3 weeks, (FIG. 5b,
lanes 1 and 2), SP1 monomer as well as non specific proteins were
removed by heat treatment for 30 mM, digested by protease
(alcalase, 10,000: dilution, FIG. 5b) and dialyzed (FIG. 5b, lane
4). MW=molecular weight markers (upper band=SP1 complex, lower
band=SP1 monomer). Note the shift from uncomplexed lower molecular
weight forms (FIG. 5a, lanes 2 and 4; FIG. 5b, lanes 1 and 2) to
higher molecular weight, oligomeric forms (FIG. 5b, lanes 3 and
4);
[0063] FIGS. 6a-6c shows the purification of recombinant SP1 from
the crude, heat-resistant extract of recombinant cells, using both
Ion Exchange (FIG. 6a) and Hydrophobic Interaction Chromatography
(FIG. 6b). FIG. 6c is a PAGE analysis of the crude recombinant cell
extract (lane 1) and heat resistant fraction (lane 2), compared to
the purified product of separation on a Source-Q hydrophobic
interaction column;
[0064] FIG. 7 shows the characterization of pure SP1, eluting as a
single peak on both gel filtration HPLC (TSK300 column) (upper) and
reverse phase HPLC (C-18 column)(lower);
[0065] FIGS. 8a-8c are a graphic representation illustrating a
hypothetical model of drug complex formation and controlled release
by the Cys2 SP1 variant. FIG. 8a shows a model for redox-dependent
opening and closing of the central cavity of the Cys2 variant,
dependent on a dynamic equilibrium between free thiols and
disulfide bonds: reducing agents shift the equilibrium towards the
free thiols and oxidizing reagents shift the equilibrium towards
the disulfide bond. FIG. 8a is a SDS-PAGE illustrating the
predominance of Cys 2 SP1 monomers under reducing conditions
(boiling for 10 min in LSB+2% b-mercaptoethanol) (lane 1), and Cys
2 SP1 dimers under non-reducing conditions (lane 2). FIG. 8c is a
graphic representation illustrating the redox-dependent drug
complex formation by the Cys2 SP1 variant.
[0066] FIG. 9 is a histogram showing redox-dependent complex
formation with small molecules by Cys2 SP1. Pure Cys 2 mutant (1.5
mg/ml in Phosphate buffered saline, pH=7.5 (PBS)) was incubated for
2 hours at room temp, with 1 mM fluorescein-amine with or without
glutathione (reduced form, GSH 3 mM). Binding reaction was stopped
by adding hydrogen peroxide (0.01%), followed by ultra filtration
(using a 30 kD cutoff filter) and extensive wash. Absorption
analysis was conducted at both 278 and 492 nm, and the results
expressed as the 492/278 ratio. Note that in the absence of the
reducing agent (GSH), retention of the flourescein-amine is
negligible;
[0067] FIG. 10 is a graphic representation of the redox-dependent
fluorescein amine complex formation by Cys2 SP1, compared to the
recombinant SP1. Fluorescein-amine was incubated with pure SP1 or
Cys2 SP1 mutant (1.5 mg/ml in PBS pH=7.5) in the absence or
presence of DTT (10 mM). Binding reaction was stopped by adding
hydrogen peroxide (0.01%), followed by ultra filtration (using 30
kD cutoff filter) and extensive wash. Samples were analyzed by Gel
filtration HPLC, and detected at both 228 and 490 nm. Calculated
data is shown on the right. Note the superior retention of the
fluorescein-amine by Cys2 SP1 mutant under reducing conditions (10
mM DTT);
[0068] FIG. 11 is a histogram illustrating the concentration
dependent complex formation with fluorescein-amine by Cys2 SP1.
Fluorescien-amine (10, 33, 100 or 333 mM) was incubated with Cys 2
SP1 or pure wild type SP1 in the presence of 10 mM DTT, and the
bound and unbound fractions analyzed by ultra-filtration and HPLC
as in FIG. 10. Note the superior binding by Cys2 SP1 of high
concentrations of Cys2 SP1;
[0069] FIG. 12 is a histogram showing the superior, redox-dependent
complex formation with Doxorubicin by Cys2 SP1. Pure wild type SP1
or Cys2 SP1 mutant (1.5 mg/ml in 20 mM Sodium Phosphate buffered,
pH=6.8) were incubated overnight at room temp, with DOX 1 mg/ml in
the presence of DTT (10 mM), with gentle rolling. Binding reaction
was stopped by adding hydrogen peroxide (0.01%), followed by ultra
filtration (using 30 kD cutoff filter) and extensive wash until
follow-through become colorless. Optical density was measured at
both 278 and 477 nm using nanodrop spectrophotometer. Note the
dramatic effect of the reducing agent on drug binding by the Cys2
SP1, compared to the absence of effect on the wild type SP1.
[0070] FIG. 13 is a histogram illustrating the redox-dependent
release of Doxorubicin by Cys2 SP1. Doxorubicin (DOX) was complexed
within Cys2 SP1 as described in FIGS. 9-12 above. Release of the
drug in the presence of 0, 2 and 20 mM GSH was measured by
ultrafiltration and size exclusion HPLC analysis (detection at 228
and 475 nm);
[0071] FIG. 14 is a histogram illustrating the effect of oxidation
on DOX complex formation by Cys2 SP1. Doxorubicin (DOX) was reacted
with Cys2 SP1 and wild type SP1 as described in FIGS. 9-13 above.
Oxidized protein indicates exposure to excess of H.sub.2O.sub.2
prior to treatment with GSH. DOX complex formation was measured by
ultrafiltration and size exclusion HPLC analysis (detection at 228
and 475 nm). Right hand panel shows the detection of bound and free
DOX on RP-HPLC, measured at 477 nm. 1=the retentate; 2=the flow
through; and 3=a DOX standard 20 .mu.g/ml.
[0072] FIG. 15 is a photograph of a SDS PAGE fluorescence analysis
characterizing the SP1-DOX complex. DOX was complexed with Cys2 SP1
variant under standard conditions (see FIG. 11), and separated by
SDS-PAGE, fixed, washed, and the DOX visualized by scanning with a
fluorescence imager (FUJIFILM FLA-500, FUJI, Japan), at 473 nm
using the green filter. Coomassie staining was used to compare
protein content of samples. Note that DOX remained tightly
complexed with all forms (complex, dimer and monomer) of the Cys 2
SP1 variant even under extreme conditions (SDS-PAGE gel);
[0073] FIG. 16 is a photograph of a fluorescent PAGE analysis
illustrating the stability of the DOX-SP1 complex upon exposure to
heat, reduction and serum. SP1 and DOX were reacted to form
complexes as described in FIGS. 9-14 above. Samples received either
heat treatment (30 minutes at 85.degree. C.) (lanes 1-4), protease
treatment (Alcalase diluted 1:000, 30 mM at 45.degree. C.)(lanes
1-3 and 5), with or without diluted mouse serum (1:10) (lanes 1 and
2). For SDS-PAGE analysis, all samples were boiled for 10 minutes
in buffer (LSB) with 2% beta-mercaptoethanol. Note the superior
resistance of the SP1-DOX complex to denaturing and proteolytic
conditions.
[0074] FIG. 17 is a photograph of a fluorescent PAGE analysis
illustrating effective complex formation with DOX by Cys2 and SP1
fusion proteins. Recombinant wild type (lanes 1 and 2), Cys2 (lanes
3 and 4), and SP1 fusion proteins having additional N-terminal
tumor specific peptide RGD (CRGD, SEQ ID NO:151; fusion protein is
SEQ ID NO: 5) (lanes 5 and 6), or RGD in reverse order (RGDC, SEQ
ID NO: 152; fusion protein is SEQ ID NO: 6) (lanes 7 and 8) were
reacted with DOX to form complex as described in FIGS. 9-14 above,
and then separated on SDS-PAGE with (lanes 2, 4, 6 and 8) or
without (lanes 1, 3, 5 and 7) denaturation by boiling in LSB. Note
the strong fluorescence in the high molecular weight complexes
(unboiled samples, lanes 1, 3, 5 and 7), indicating effective
binding of the drug by all variant SP1 and to much lower extent by
wild type;
[0075] FIG. 18 is photograph showing the resistance of SP1 high
molecular weight oligomeric complex to organic solvents. Wild type
SP1 (lanes 1-3) or Cys2 SP1 (lanes 4-6) (1 mg/ml in 10 mM sodium
phosphate pH 7) were lyophilized and re-suspended in buffer (lanes
1 and 4) or solvent (10 minutes incubation time) (lanes 2 and
5=methanol; lanes 3 and 6=hexane). Samples were resuspended in
water and analyzed for the presence of high molecular weight
oligomeric complexes by SDS-PAGE. Note the persistence of
oligomeric complexes under all conditions, and that the Cys2 SP1
variant is even more resistant than the wild type;
[0076] FIG. 19 is an HPLC analysis showing the solublization of
Paclitaxel (PTX) by complex with SP1. Purified recombinant,
freeze-dried wild type-SP1 was mixed with Paclitaxel solution (0.1
ml, dissolved in acetone:hexane (1:2) 0.25 mg/ml), sonicated for 20
minutes, and solvents evaporated by speed vacuum. Following
dissolving in water, additional sonication and vortex mixing, 50
.mu.l samples were analyzed by RP HPLC. Top panel shows the HPLC
peaks (at 225 nm) (SP1=black; SP1-PTX=red; PTX=blue; PTX in
H.sub.2O=magenta). Lower panel shows the results of ultrafiltration
(at 30 kD) of the complexed SP1-PXT. Note the high percentage of
PTX retained in solution by the SP1-PXT complex;
[0077] FIG. 20 is an HPLC analysis showing efficient ethanol
extraction of PTX from the SP1-PTX complex. SP1-PTX complex
prepared as in FIG. 19 above was precipitated (red line) and then
extracted (yellow line) with 80% ethanol (2 hours at -20.degree.
C.), and then analyzed on HPLC (lower panel) (blue=untreated
complex). Note the loss of PTX with protein precipitation, and the
appearance of PTX in the extracted sample;
[0078] FIG. 21 is an HPLC analysis showing the effect of reducing
conditions on PTX extraction. SP1-PTX complex prepared as in FIG.
19 above was extracted with 0-60% ethanol (under conditions not
causing protein precipitation) in the presence (closed
square-magenta) or absence (closed triangle-blue) of 10 mM GSH, and
then analyzed on HPLC. Note the superior retention of PTX by the
oxidized complex;
[0079] FIG. 22 is an HPLC analysis showing the effects of reducing
conditions on PTX binding by SP1. SP1-PTX complex was prepared as
in FIG. 19 in the absence or presence of reducing conditions
(b-mercaptoethanol mM), separated by ultrafiltration (sterile 0.22
.mu.m filter) and HPLC as described above. Note the superior water
solubility of the complexes formed under reducing conditions;
[0080] FIG. 23 is a graph showing the complex formation of the drug
Vinblastine to SP1. Left panel-emission spectra (excitation
wavelength=286.00 nm) of pure SP1 (48 .mu.M in MES) was determined
using a fluorometer. Both native (left curves) and unfolded (6M
Guanidinum HCl) (right curves) SP1 were tested. The net effect of
Vinblastine on Tryptophan fluorescence of native protein was
calculated by subtracting the relative quenching by the unfolded
protein from those of the native protein at the respective maximal
emission wavelength (340 nm and 321 nm respectively). Note that the
tryptophan fluorescence of both the folded and unfolded SP1 protein
is quenched by Vinblastine complexing, but in the case of the
folded protein is also accompanied by a red shift);
[0081] FIGS. 24a and 24b are graphs illustrating the in-vitro
cytotoxic effects of SP1-DOX complex. FIG. 24a--HT-29 cells
cultured in 96 well microtiter plates were exposed to uncomplexed
DOX (closed oval, magenta) or SP1-DOX complex (closed triangle,
blue) prepared as described in FIGS. 9-15, at the indicated
concentrations. FIG. 24b--HT-29 cells were exposed to uncomplexed
SP1 (without DOX) (closed diamond, blue) or SP1-DOX complex (closed
triangle, yellow) at indicated concentrations. Proportion of living
cells was determined by MTT assay, and the IC.sub.50 was
calculated. Note the absence of cytotoxicity of uncomplexed SP-1
(FIG. 24b), and the equivalent IC.sub.50 values for free DOX and
for the SP1-complexed DOC;
[0082] FIGS. 25a and 25b are graphs showing the in-vitro
cytotoxicity of SP1-PTX complex compared to free PTX. SP1-PTX
complex was prepared according to FIGS. 19-22 above. HT-29 cells
were prepared as in FIG. 24 above. FIG. 24a shows the IC.sub.50 of
HT-29 cells exposed to SP1-PTX and free PTX (in DMSO). The cells
were exposed to free PTX (closed circles, green) or SP1-PTX complex
(closed triangle, red), at the indicated concentrations. FIG. 24b
shows the IC.sub.50 of HT-29 cells exposed to uncomplexed SP1
(close triangle, blue) or SP1-PTX complex (closed triangle, red) at
indicated concentrations. Note the absence of cytotoxicity of the
uncomplexed SP1, and the similar IC.sub.50 values for both free PTX
and SP1-PTX complex.
[0083] FIG. 26 is an immunoblot analysis illustrating the superior
pharmacodynamics and targeting of SP1 complex. C57Bl male mice
bearing the B16-F10 (B16) melanoma tumor were divided into three
groups: Group A--injected once (iv with fluoresceinamine-SP1
conjugate (10 mg/ml, 0.1 ml per mouse), n=5 mice;
[0084] Group B--injected once with unconjugated fluoresceinamine
solution (34 mM in PBS, 0.1 ml per animal); n=5 mice; and Group C
received no treatment. n=2 mice. Internal organs were harvested 24
hours post injection, and stored at 70.degree. C. Blood was
collected and left at room temperature to coagulate. Tumor extracts
and serum samples were analyzed on SDS PAGE, and proteins were
blotted onto nitrocellulose. Immunodetection of SP1 was with rabbit
anti SP1 and HRP-conjugated second antibody. Note the abundance of
SP1 in both tumors and serum at 24 hours post injection;
[0085] FIGS. 27a and 27b are histograms showing the superior
anti-tumor effects of SP1 complexed DOX compared to uncomplexed
DOX. CD1 nude mice bearing human LS 147T colon cancer (one million
cells per animal) subcutaneous xenografted tumors were divided into
two groups (n=6), and received either SP1-Dox (50 mg/Kg in PBS,
about 0.5 mg DOX equivalent/Kg) or PBS (FIG. 27a), or uncomplexed
DOX (3 mg/Kg) or PBS (FIG. 27b) alone injected intravenously into
the tail vein twice per week for four weeks. Tumors were removed
and weighed 35 days post engraftment. Note the significantly
greater anti-tumor effectiveness of the complexed SP1-DOX, as
compared to free DOX;
[0086] FIGS. 28a and 28b are histograms showing the significant
reduction in side effects with SP1-complexed DOX treatment,
compared to free DOX. CD1 nude mice bearing s.c. xenografted human
LS147T colon cancer tumors were treated with intravenous
SP1-complexed DOC (FIG. 28a) or free, uncomplexed DOC (FIG. 28b) as
described in FIGS. 27a and 27b hereinbove. PBS was injected to the
controls. Animals were weighed before sacrifice (35 days post tumor
injection). Note the severe weight loss with uncomplexed, free DOX,
compared to the negligible weight loss in mice receiving
SP1-complexed DOX;
[0087] FIG. 29 is a graph showing the detection of SP1-DOX complex
by size-exclusion HPLC analysis. SP1-DOX complex is detectable at
both 278 nm (characteristic for SP1) and 475 nm (characteristic for
DOX).
[0088] FIG. 30 shows a typical SP1 standard curve on size exclusion
chromatography (size exclusion HPLC) at 278 nm. SP1 is eluted from
the column (TSK G3000 SWXL, Tosohaas) after 7 min and is detected
at 278 nm only. Inset shows the quantitative detection of the SP1
over a range of concentrations.
[0089] FIG. 31 shows chromatograms of size exclusion chromatography
(size exclusion HPLC) of FA standard profile at 490 nm. In contrast
with free FA, which is eluted from the column in a distinctive
peak, DOX is not eluted in a distinctive peak (FIG. 29). Inset
shows the quantitative detection of the FA over a range of
concentrations.
[0090] FIG. 32 shows the standard profiles of Cys2 SP1 (determined
at both 278 (left panel) and 225 (right panel) nm) on RP-HPLC.
Insets show the quantitative detection of the Cys2 SP1 over a range
of concentrations.
[0091] FIG. 33 shows the standard profile of DOX (determined at 477
nm) on RP-HPLC. Inset show the quantitative detection of the DOX
over a range of concentrations.
[0092] FIG. 34 shows the standard profile for PTX (determined at
225 nm) on RP-HPLC. Inset show the quantitative detection of the
PTX over a range of concentrations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0093] The present invention is of SP1 and SP1 variant polypeptides
and polynucleotides encoding same capable of forming molecular
complexes, which can be used for nanoparticles and selective
complexing and release of substances.
[0094] Specifically, the present invention can be used to deliver,
stabilize and solubilize therapeutic, diagnostic, cosmetic,
conductive and semi-conductive agents and the like. The homo- and
hetero-oligomeric complex formation characteristic of SP1 and SP1
variant polypeptides of the present invention can also be used to
provide engineered self-assembling nanoparticles and
nanostructures. Further, SP1 variants having a wide variety of
complex-forming modifications (such as disulfide and other peptide
linkages, carbohydrate, nucleic acids, etc, and combinations
thereof) can be designed, producing large and varied possibilities
for controlled complex and dissociation of hetero- and homo
oligomeric SP1 structures, and of SP1 polypeptides complex
formation with, and release of small molecules, drugs, agents,
nanoparticles and the like. Additional aspects and applications of
the invention are further discussed below.
[0095] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0096] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0097] SP1 polypeptide is an exceptionally stable polypeptide,
forming hetero- and homo-oligomers which are resistant to
denaturation by heat and most chemical denaturants, resistant to
protease digestion, and capable of stabilizing molecular
interactions and forming three dimensional structures (Dgany et al,
JBC, 2004; 279:51516-23, and U.S. patent application Ser. No.
10/233,409 to Wang et al)
[0098] The present inventors have previously uncovered SP1 proteins
fused to other protein or non-protein molecules, for enhancement of
binding properties of binding molecules, for stabilization of the
fused molecules (such as enzymes) and for enhancement or alteration
of immunological properties of the fused molecules (U.S. patent
application Ser. No. 10/233,409 to Wang et al.). SP1 fusion
proteins, as disclosed in U.S. patent application Ser. No.
10/233,409, comprise recombinant SP1 molecules having additional
polypeptide sequences added by genetic engineering techniques, and
SP1 molecules having additional non-protein moieties added by
chemical means, such as cross linking. The present inventors have
further disclosed the therapeutic use of SP1 proteins for
strengthening skin, hair, nails, etc.
[0099] However, Wang et al. do not teach, nor imply native SP1, or
SP1 variant polypeptides capable of controlled release of agents
(therapeutic, cosmetic, diagnostic, conductive, etc) in molecular
association therewith, the use thereof as carriers or the use of
self-assembling SP1 monomers for the production of
nanostructures.
[0100] While reducing the present invention to practice, novel SP1
variants were produced through laborious experimentation and drug
design which are capable of hetero- and homo-oligomer formation,
and formation of reversible molecular complexes with a variety of
substances and molecules. The controllable nature of the SP1
molecular complexes of the present invention makes the SP1
polypeptides exceptionally useful as carriers for drugs, cosmetics,
conductors and other small molecules. Further, specific moieties
can be incorporated to add target recognition capabilities to the
SP1 polypeptide, enhancing the specificity and efficacy SP1 as a
drug carrier. Further, while reducing the present invention to
practice, it was surprisingly uncovered that native SP1 and
modified SP1 variants can self assemble to produce defined
nanostructures, in a controlled and predetermined manner. Such
nanostructures can be used for engineering, electrical and other
nano-technological applications.
[0101] Thus, according to one aspect of the present invention there
is provided an isolated polypeptide comprising an amino acid
sequence of an SP1 polypeptide, the amino acid sequence being
modified to be in a reversible molecular association with a
substance.
[0102] As used herein the phrase "molecular association" refers to
a chemical association or a physical association or both, which
takes place on a molecular level. For example, an association can
be a covalent bond, a non-covalent bond, a hydrophobic interaction,
etc.
[0103] A "reversible association," as defined herein, is an
association wherein the components can return to an original,
pre-association, state, and reassociate, depending on the specific
conditions. Preferably such association and reassociation does not
include the formation and cleavage of peptide bonds. For example, a
reversible association of the components of a SP1-therapeutic agent
complex of the invention can disassociate and thereby return to
original and distinct therapeutic agent and SP1 polypeptide
components.
[0104] Types of reversible molecular associations suitable for use
in the present invention are associations selected from the group
consisting of electrostatic bonding, hydrogen bonding, van der
Waals forces, ionic interaction or donor/acceptor bonding. The
reversible association can be mediated by one or more associations
between the substance and the SP1 polypeptide. For example, the
reversible association can include a combination of hydrogen
bonding and ionic bonding between the complexing substance and the
SP1 polypeptide. Additionally, or alternatively, the reversible
association can be in combination with, for example, covalent or
other noncovalent interactions between components, such as between
a substance and an SP1 polypeptide.
[0105] As used herein the phrase "SP1 polypeptide" refers to a
protein having at least one of the following characteristic
properties: boiling stability, protease stability or chaperone-like
activity, from aspen and other plants, and belonging to the family
of SP1 and SP1-like proteins (see SEQ ID NOs:123-148).
[0106] SP1 polypeptides are characterized by at least one of the
following distinctive properties: stability, chaperone-like
activity and excellent resistance to denaturing factors. SP1
polypeptides also share some regions of conserved sequence
homology. Members of the SP1 family are preferably boiling and
detergent stable proteins at least 65% homologous to SEQ ID NO:1,
the boiling and detergent stable proteins having a chaperone-like
activity and being capable of forming stable dimers. Yet more
preferably, SP1 polypeptides have at least one conserved amino acid
sequence in at least one region corresponding to amino acids 9-11,
44-46 and/or 65-73, of SEQ ID NO:1, as determined using a Best Fit
algorithm of GCG, Wisconsin Package Version 9.1, using a plurality
of 10.00, a threshold of 4, average weight of 1.00, average match
of 2.91 and average mismatch of minus 2.00. Most preferably, the
SP1 polypeptide has conserved consensus sequences: "HAFESTFES"
(65-73, SEQ ID NO:1), "VKH" (9-11, SEQ ID NO:1) and "KSF" (44-46,
SEQ ID NO:1). Most preferably, "wild-type" SP1 is the stress
related SP1 protein from aspen (SEQ ID NO:1), as disclosed by Wang
et al (U.S. patent application Ser. No. 10/233,409, filed Sep. 4,
2002, which is a Continuation in Part of PCT IL 02/00174, filed
Mar. 5, 2002, both of which are incorporated by reference as if
fully set forth herein.).
[0107] In a preferred embodiment, the SP1 protein is 70%, more
preferably 75%, yet more preferably 80%, more preferably 85%, more
preferably 90%, preferably 95%, and most preferably 100% homologous
to SEQ ID NO: 1.
[0108] As used herein the phrase "denaturant-stable" refers to
major (above 50%) structural oligomeric stability following a
denaturation treatment in aqueous solution. A denaturation
treatment can include boiling and exposure to a chemical
denaturant, such as, a detergent (e.g., SDS), urea, or
guanidinium-HCl.
[0109] As used herein, the phrase "boiling stable" refers to major
(above 50%) structural oligomeric stability following treatment at
substantially 100.degree. C. in aqueous solution for at least 10
minutes, as determined by a size fractionation assay.
[0110] As used herein, the phrase "detergent stable" refers to
major (above 50%) structural oligomeric stability of an oligomeric
protein following treatment in aqueous solution containing 1/2,000
molar ratio (monomer:SDS), as determined by a size fractionation
assay.
[0111] As used herein in the specification and in the claims
section that follows, the phrase "protease resistant" refers to
major (above 50%) stability following treatment in aqueous solution
containing 50 .mu.g per ml proteinase K for at least 60 minutes at
37.degree. C.
[0112] As used herein, the phrase "chaperone-like activity" refers
to the ability to mediate native folding and native oligomerization
of proteins, to prevent the formation of incorrect protein
structures, to unscramble existing incorrect protein structures and
to limit stress-related damage by inhibiting incorrect interactions
that could occur between partially denatured proteins or their
domains.
[0113] As mentioned hereinabove, the amino acid sequence of the
isolated polypeptide of the present invention is modified to render
it capable of forming reversible molecular associations with other
molecules. Interestingly and surprisingly polypeptides of this
aspect of the present invention retain the above-mentioned
activities of native SP1 polypeptide such as ability of forming
oligomers that are heat-stable and denaturant- and
protease-resistant (see Example 2, FIGS. 4-6 hereinbelow).
[0114] Modified SP1 polypeptides of the present invention are
designed to have a novel activity of interest (e.g, reversible
association with a substance, cellular recognition, etc) which is
not featured in wild type SP1 while still maintaining at least one
of the above SP1 activities. Assays for testing such polypeptides
are described hereinabove.
[0115] As used herein, the term "modified amino acid sequence"
refers to an amino acid sequence having any deviation from the
amino acid sequence of a native SP1 polypeptide, as described
hereinabove. Modifications of SP1 polypeptide include, but are not
limited to substitution of amino acids, addition of amino acids,
deletion of amino acids, addition of di-, tri-, oligo- or
polypeptides to the SP1 polypeptide, transposition of one or more
amino acids from one portion of the amino acid sequence to another
portion of the sequence, alterations of existing amino acids, such
as cross-linking or elimination of portions of the side chains,
addition of linkers, truncation of the amino acid sequence,
addition of non-peptide moieties such as carbohydrates, lipids,
nucleic acids and the like, introduction of substances having
magnetic properties, etc. Examples of specific modifications are
described in detail hereinbelow.
[0116] Modified SP1 variant polypeptides can be modified to impart
specific properties to the SP1 variant, thereby rendering the
molecular complexing with, and release of other substances more
efficient and controllable, and adaptable to specific conditions.
Thus, for example, addition of thiol (S--H) groups can produce SP1
variants having redox-sensitive molecular complex formation,
between SP1 and complexing substances and between SP1 monomer,
dimers, trimers etc. This can be useful for designing drug carriers
improving the specificity of dosing and drug regimen. Further,
modification of SP1 polypeptide amino acid sequence by addition of
oligo- or polypeptide sequences capable of reversibly binding
inorganic molecules such as metals and other ions can be useful for
forming conductive compositions and altering the magnetic
properties of molecular complexes formed by SP1 polypeptides.
Modifications of the SP1 amino acid sequence altering interactions
between the oligomer subunits, such as the dimer-dimer or
monomer-monomer interactions, can serve to stabilize, or
destabilize oligomer conformation, rendering the SP1 variants
potentially more or less resistant to the chemical environment.
Such increased, or decreased stability can be designed to affect
the properties of modified SP1 variants as a carrier, for example,
as a drug carrier. Such modifications in the subunit-subunit
interactions of the SP1 variant can also be used to design and
control the properties of SP1-based nanostructures.
[0117] It will be appreciated that the SP1-complex formation can
also be based on intermolecular crosslinking mechanisms to bridge
between two neighboring subunits. Examples include thiol-, amine,
carboxyl and hydroxyl reactive crosslinking reagents. In such cases
the controlled release mechanism can also be based on cleavage of
the crosslinking by enzymatic activity as well as by using
cleavable crosslinkers.
[0118] As mentioned above, the SP1 amino acid sequence can be
modified to include additional peptide moieties. Thus,
alternatively and additionally, the SP1 polypeptide can be modified
to include at least one recognition sequence. Such recognition
sequences include, but are not limited to target recognition
sequences such as cell surface recognition sequences, specific
ligands such as receptor binding ligands, antibodies or portions
thereof such as antibody binding sites, organ- and tissue-specific
recognition sequences, developmental stage-specific recognition
sequences, species- and sex-specific recognition sequences, and
recognition sequences correlating with specific diseases or
conditions. A non-limiting list of suitable recognition sequences
includes tumor surface specific peptides KNGPWYAYTGRO (SEQ ID NO:
31), NWAVWXKR (SEQ ID NO: 32), YXXEDLRRR (SEQ ID NO: 33). XXPVDHGL
(SEQ ID NO: 34), LVRSTGQFV (SEQ ID NO: 35), LVSPSGSWT (SEQ ID NO:
36), ALRPSGEWL (SEQ ID NO: 37), AIMASGQWL (SEQ ID NO: 38),
QILASGRWL (SEQ ID NO: 39), RRPSHAMAR (SEQ ID NO: 40), DNNRPANSM
(SEQ ID NO: 41), LQDRLRFAT (SEQ ID NO: 42), PLSGDKSST (SEQ ID NO:
43), FDDARL (SEQ ID NO: 44), FSDARL (SEQ ID NO: 45), FSDMRL (SEQ ID
NO: 46), FVDVRL (SEQ ID NO: 47), FTDIRL (SEQ ID NO: 48), FNDYRL
(SEQ ID NO: 49), FSDTRL (SEQ ID NO: 50), PIHYIF (SEQ ID NO: 51),
YIHYIF (SEQ ID NO: 52), RIHYIF (SEQ ID NO: 53), IEILQAR (SEQ ID NO:
54), CVFXXXYXXC (SEQ ID NO: 55), CXFXXXYXYLMC (SEQ ID NO: 56),
CVXYCXXXXXCYVC (SEQ ID NO: 57), CVXYCXXXXCWXC (SEQ ID NO: 58),
DPRATPGS (SEQ ID NO: 59), HLQLQPWYPQIS (SEQ ID NO: 60),
VPVWMEPAYQRFL (SEQ ID NO: 61), TSPLNIHNGQKI (SEQ ID NO: 62).
Suitable tumor vascular peptides for use with the modified SP1
polypeptide of the present invention include, but are not limited
to CDCRGDCFC (RGD-4C) (SEQ ID NO: 63), ACXDCRGDCFCG (SEQ ID NO:
64), CNGRCVSGCAGRC (SEQ ID NO: 65), CVCNGRMEC (SEQ ID NO: 66),
NGRAHA (SEQ ID NO: 67), TAASGVRSMH (SEQ ID NO: 68), LTLRWVGLMS (SEQ
ID NO: 69), CGSLVRC (SEQ ID NO: 70), CGLSDSC (SEQ ID NO: 71),
NRSLKRISNKRIRRK (SEQ ID NO: 72), LRIKRKRRKRKKTRK (SEQ ID NO: 73),
NRSTHI (SEQ ID NO: 74), SMSIARL (SEQ ID NO: 73), VSFLEYR (SEQ ID
NO: 76), CPGPEGAGC (SEQ ID NO: 77), ATWLPPR (SEQ ID NO: 78), RRKRRR
(SEQ ID NO: 79), ASSSYPLIHWRPWAR (SEQ ID NO: 80), CTTHWGFTLC (SEQ
ID NO: 81).
[0119] As mentioned hereinabove, the SP1 polypeptide of the
invention can be modified to form a reversible molecular
association with a substance. Substances suitable for forming a
reversible complex with the polypeptide of the present invention
include, but are not limited to, a therapeutic, diagnostic or
cosmetic agent. Suitable therapeutic, diagnostic or cosmetic agents
include, but are not limited to a polypeptide agent, a nucleic acid
agent, a lipid agent, a carbohydrate agent, and a small
molecule.
[0120] Therapeutic agents suitable for use with the polypeptide of
the present invention include, but are not limited to drugs and
biologically active molecules such as anti-inflammatory drugs and
anti-cancer (oncology) drugs). Anti inflammatory drugs that can be
complexed in molecular association with SP1 of the present
invention include but are not limited to Alclofenac; Alclometasone
Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal;
Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra;
Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac;
Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole;
Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone
Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;
Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac
Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone
Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide;
Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole;
Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac;
Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort;
Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin
Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone;
Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen;
Furobufen; Halcinonide; Halobetasol Propionate; Halopredone
Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen
Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen;
Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam;
Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol
Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone
Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;
Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;
Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;
Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;
Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate;
Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine;
Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;
Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;
Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone;
Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin;
Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium;
Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol
Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate;
Zidometacin; Zomepirac Sodium.
[0121] Anti-cancer drugs suitable for complexing and use with the
polypeptide of the present invention include, but are not limited
to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;
Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin;
Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;
Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;
Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene
Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;
Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin
Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;
Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide;
Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride;
Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;
Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;
Droloxifene; Droloxifene Citrate; Dromostanolone Propionate;
Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin;
Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride;
Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine
Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate;
Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide;
Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorocitabine;
Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine
Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;
Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon
Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; Interferon
Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide
Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;
Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;
Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol
Acetate; Melengestrol Acetate; Melphalan; Menogaril;
Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;
Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;
Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman;
Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin;
Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;
Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin;
Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol;
Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;
Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;
Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride;
Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate;
Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole
Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin;
Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine
Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine
Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine
Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.
Additional antineoplastic agents include those disclosed in Chapter
52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner),
and the introduction thereto, 1202-1263, of Goodman and Gilman's
"The Pharmacological Basis of Therapeutics", Eighth Edition, 1990,
McGraw-Hill, Inc. (Health Professions Division).
[0122] Diagnostic substances that can be used with the SP1
polypeptides of the present invention include, but are not limited
to radioactive substances, light emitting substances,
radio-frequency transmitters and receivers, magnetic substances,
pigmented substances, chemically active substances such as
oxidizing, reducing, cross-linking, etc agents, FRET-pairs, QUANTUM
dots, biochemical substances capable of molecular recognition such
as nucleic acids, antibodies, etc, biologically active species such
as enzymes, and the like.
[0123] According to another aspect of the present invention, the
substance is a conductive or semi conductive agent. As used herein,
a "conductive agent" refers to an agent capable of moving an
electrically charged particle through a transmission medium.
Examples of conductive agents include metals and many ionic
substances. As used herein, a "semiconductive agent" refers to an
agent having insulating properties, which can also, under given
conditions, move an electrically charged particle through a
transmission medium. Semiconductive agents behave as an insulator
at very low temperatures, and have an appreciable electrical
conductivity at room temperature although much lower conductivity
than a conductor. Commonly used semiconducting materials are
silicon, germanium, gallium arsenide, indium phosphide, and mercury
cadmium telluride.
[0124] Modifications shown suitable for complex formation with
conductive agents such as metal and other inorganic ionic
substances include the 6H his tag for complex formation with Nickel
and other metal ions (SEQ ID NO:122). Table 1 hereinbelow is a
non-limiting list of additional peptides forming complexes with
inorganic ionic substances that are suitable for modification of
the SP1 polypeptide (adapted from Sarikaya et al., Ann Rev Mater
Res 2004; 34:373-408).
TABLE-US-00001 TABLE 1 A list of inorganic-binding polypeptides
selected by phage display (PD) and cell surface display (CSD)
Metals and SEQ metal Sequence origin and oxides reference Sequences
ID Au CSD outer membrane MHGKTQATSGTIQS 82 SKTSLGCQKPLYMGREMRML 83
QATSEKLVRGMEGASLHPAKT 84 Pt PD: constrained 7aa (S. DRTSTWR 85
Dincer, C. Tamerler & M. Sarikaya, unpublished) QSVTSTK 86
SSSHLNK 87 Pd PD: constrained 7aa (S. SVTQNKY 88 Dincer, C.
Tamerler & M. Sarikaya, unpublished) SPHPGPY 89 HAPTPML 90 Ag
PD: 12 aa AYSSGAPPMPPF 91 unconstrained NPSSLFRYLPSD 92
SLATQPPRTPPV 93 SiO.sub.2 PD:12 aa unconstrained MSPHPHPRHHHT 94
RGRRRRLSCRLL 95 KPSHHHHHTGAN 96 PD:12 aa unconstrained YSDQPTQSSQRP
97 (D. Sahin, C. Tamerler & M. Sarikaya, unpublished)
TYHSSQLQRPPL 98 SPLSIAASSPWP 99 Silaffins SSKKSGSYSGYSTKKSGSRRIL
100 SSKKSGSYSGSKGSKRRIL 101 SSKKSGSYSGSKGSKRRNL 102 Silicatein
.alpha. SSRCSSSS 103 ZnO CSD: flagella) VRTRDDARTHRK 104 CSD:
fimbria) PASRVEKNGVRR 105 NTRMTARQHRSANHKSTQRA 106 YDSRSMRPH 107
Cu2O CSD: flagella) RHTDGLRRIAAR 108 RTRRQGGDVSRD 109 RPRRSAARGSEG
110 Zeolites CSD: outer membrane) VKTQATSREEPPRLPSKHRPG 111
MDHGKYRQKQATPG 112 CaCO.sub.3 PD: 12 aa HTQNMRMYEPWFG 113
unconstrained) DVFSSFNLKHMRG 114 Cr.sub.2O.sub.3 CSD: fimbria)
VVRPKAATN 115 RIRHRLVGQ 116 Fe.sub.2O.sub.3 CSD: outer membrane)
RRTVKHHVN 117 GaAs PD: 12 aa AQNPSDNNTHT 118 unconstrained)
RLELAIPLQGSG 119 TPPRPIQYNHTS 120 ZnS PD: 7aa constrained) NNPMHQN
121
[0125] Thus for example, the modified SP1 polypeptide of the
present invention may be in reversible molecular association with a
variety of therapeutic, cosmetic, diagnostic, conductive, etc
substances.
[0126] As mentioned hereinabove, modified SP1 variant polypeptides
can be modified to impart specific properties to the SP1 variant,
thereby rendering the molecular complexing with, and release of
other substances more efficient and controllable, and adaptable to
specific conditions. It will be appreciated, that through intensive
investigation into the properties of the SP1 polypeptide and
oligomer complex, certain sequences of the SP1 polypeptide have
been associated with one or more of the properties characteristic
of the SP1 family (see, for example, Dgany et al, JBC 2004
279:51516-523). Thus, modifications within specific regions of the
SP1 polypeptide can be introduced, which can in turn result in
desired alterations in the properties of the SP1 variants, such as
modes of molecular association, oligomer formation, etc. Dgany et
al (JBC 2004 279:51516-523) have identified a number of
structurally significant regions in the SP1 polypeptide:
[0127] The SP1 monomer protein chain has an .alpha.- and
.beta.-folding with three .alpha.-helices, H1 (residues 23-39), H2a
(residues 74-81), and H2b (residues 84-93), and a .beta.-sheet
formed by four antiparallel .beta.-strands, B3 (residues 9-17), B1
(residues 45-50), B2 (residues 65-71), and B4 (residues 97-108).
The N-terminal segment points toward the solvent and is mobile. A
long, largely unstructured loop is formed by residues 51-64, which
may be involved in dimer contacts. Helices H1 and H2 define an
external convex surface forming a central cavity with the opposing
.beta.-sheet. Hence, for example, modifications within the long
loop may effect the stability (enhance or decrease) of dimer-dimer
contacts, and oligomer formation, resulting in, for example, an SP1
variant drug carrier having a longer or shortened half-life after
administration.
[0128] The dimer appears to be the smallest stable SP1 unit. The
two molecules in the dimer are related by a 2-fold axis parallel to
helix H1 and .beta.-strands B3 and B4. The outer surface of the
.beta.-sheets of the two molecules forms a .beta. barrel-like
structure, defining a central pore. Modifications of this region of
the SP1-polypeptide may affect the internal hydrophobic molecular
environment, in turn either enhancing or decreasing the ability to
complex with hydrophobic molecules.
[0129] In the oligomeric dodecamer, the interdimer contacts
predominantly involve hydrophilic side chains and charged groups or
are mediated by water molecules. These contacts take place mainly
along the B1, H1, and the N-terminal tails. Table 2 shows a
non-limiting list of novel SP1 variants produced having a modified
amino acid sequence, including modifications in specific regions of
the SP1 polypeptide described by Dgany et al. (JBC 2004
279:51516-523).
TABLE-US-00002 TABLE 2 Characterization of SP1 mutant proteins
Location .beta.-ME based on SP1 Resistance dependent crystal
Complex Protease to 2M Heat Dimer SEQ ID structure
Modification.sup.1 Formation.sup.3 Resistance.sup.4 GHCl.sup.5
stability.sup.6 Tm.sup.7 formation.sup.8 NO: wild type + + + + 107
- 1 N-terminus .DELTA.2-6 + + + + 104 - 2 modification Cys2 + + ND
+ ND + 3 HH2 + + ND + ND - 4 CRGD2 + + ND + ND + 5 RGDC2 + + ND +
ND + 6 6H2 + + + + 109 - 7 .DELTA.2-6 + + ND + ND - 8 His2
.DELTA.2-7 + + ND + ND + 9 Cys2 Loop1 E20K 9 NA NA NA NA NA 10
modification Cys2 10+ + ND + ND + 11 (residues 18-22) K18R
Gly19.sup.2 Dimer-dimer R23A + + + + 108 - 12 interaction D27A + +
+ + N.D - 13 region I30A + + + - 98 - 14 modification N31A + + + +
110 - 15 T34A + + + + 114 - 16 D38A + + + + 113 - 17 Loop 2
.DELTA.2-6 + + + + ND - 18 modification I40C (residues 40-44)
Monomer- E68A + + + + 105.8 - 19 monomer region interactions Loop 4
.DELTA.2-6 10 + + + ND - 20 modification E72C (residues 72-73)
.DELTA.2-6 11 + + + ND - 21 S73C The external .DELTA.2-6 10+ + + +
ND - 22 perimeters of L81C the dodecamerring Destabilization F106A
+ + - - 75 - 23 of 6XH2 Dimmer-dimer Y108A + + - - 68 - 24
interactions N31A ? NA NA NA NA NA 25 Y108A 9 6XH2 T50A NA NA NA NA
NA 26 I52A 9 6XH2 F106A NA NA NA NA NA 27 Y108A 9 6XH2 S73A + + + +
NA - 28 S75A 6XH2 D38A + + + + 112 - 29 S75A 6XH2 N31A + + + - 96.6
- 30 T34A 6XH2 ND not determined; NA not applicable .sup.1Standard
nomenclature for mutation (amino acid position using wild type
sequence including first Methionine residue). .sup.2Insertion of
Cys residue in position 2 and of glycine residue in position 19
K18R (Cys2loop1RGd). .sup.3Tested by SDS PAGE when samples are not
boiled in the application buffer Several SP1 mutants fail to form a
soluble protein during expression and form inclusion bodies (IB).
These IBs were unfolded with 0.5M Urea, and refolded by dialysis.
.sup.4Tested by SDS PAGE after either proteinase K (50 ug/ml; 30
min; .degree. C.), or alkalase ( 1/1000 dilution 60 min; 45.degree.
C.) treatment, conditions under which SP1 monomer as well as most
other proteins degrade. .sup.5Complex stability following
incubation 2M GHCl (1 h at room temp) was tested by SDS PAGE.
.sup.6Heat stable protein is defined as one that does not
precipitate after heat treatment 10 min incubation at 100.degree.
C. or 30 min incubation at 85.degree. C. .sup.7Protein melting
point was tested by DSC. .sup.8Dimer formation is tested by SDS
PAGE when samples are boiled for 10 min in the application buffer,
in the absence or presence of b-mercaptoethanol. .sup.9Inclusion
Body (IB) refolding was not tested. .sup.10Forms complex after IB
refolding. Complex assembly was confirmed by eliminating the
monomeric forms using proteinase digestion.
[0130] As shown in Table 2, for example, modifications including
addition of amino acids having thiol groups characteristically have
redox-dependent (.beta.-ME) dimer formation and modifications in
the amino acid sequence of the N-terminus portion of the
polypeptide typically retain the ability to form oligomeric
complexes, resistance to protease digestion, heat stability and
resistance to guanidinium HCl denaturation.
[0131] Examples of modified SP1 variants such as SP1 6H (SEQ ID NO:
7), SP1 .DELTA.N (SEQ ID NO:2), Cys2 SP1 (SEQ ID NO:3), CRGD SP1
(SEQ ID NO:5) and RGDC SP1 (SEQ ID NO:6) formed homo- and
hetero-oligomeric complexes which showed characteristic stability
and resistance.
[0132] Thus, while reducing the present invention to practice, it
was uncovered that while some modified SP1 variants form boiling
and protease stable complexes, others destabilize the oligomeric
complexes. N-terminal truncated (.DELTA.N) (SEQ ID NO:2) and 6H
histidine tagged (SEQ ID NO:7) SP1 variants retained stable
oligomeric complex formation (FIG. 3, Example 2). On the other
hand, other substitutions led to the destabilizing of the complex
formation, and decreased solubility (see FIG. 5, Example 3) of the
recombinant protein.
[0133] Further, the modified SP1 variants retained the capability
to form oligomeric, high molecular weight complexes. 6H tagged (SEQ
ID NO:7) and N-terminal truncated (SEQ ID NO: 2), when dissociated
into monomers by extreme conditions, regained oligomer form in both
homo-oligomeric (.DELTA.N-.DELTA.N; and 6H 6H) and
hetero-oligomeric conformations (.DELTA.N 6H) (FIG. 5, Example
3).
[0134] Modification of the SP polypeptide amino acid sequence can
be introduced by chemical, recombinant or other means. In one
embodiment, modification of the amino acid sequence of SP1 is a
chemical modification, such as carbodiimide conjugation,
glutaraldehyde conjugation, SPDP conjugation, acylation,
glycosylation, alteration of functional groups, cross-linking of
amino acids, deletions and the like. The amino acid sequence can be
modified by addition, usually covalent, of non-peptide molecules
such as lipids, nucleic acids and carbohydrates. Other examples of
peptide modification are described herein in detail.
[0135] The term "peptide" as used herein encompasses native
peptides (either degradation products, synthetically synthesized
peptides or recombinant peptides) and peptidomimetics (typically,
synthetically synthesized peptides), as well as peptoids and
semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells. Such
modifications include, but are not limited to N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH2-NH, CH2-S, CH2-S.dbd.O,
O.dbd.C--NH, CH2-O, CH2-CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH,
backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and
are specified, for example, in Quantitative Drug Design, C.A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which
is incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0136] The SP1 polypeptides contemplated herein include, but are
not limited to, modifications to side chains, incorporation of
unnatural amino acids and/or their derivatives during peptide
synthesis and the use of crosslinkers and other methods which
impose conformational constraints on the peptides or their
analogues.
[0137] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with
2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5'-phosphate followed
by reduction with NaBH.sub.4.
[0138] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0139] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivitisation, for example, to a corresponding amide.
[0140] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0141] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0142] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0143] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids.
[0144] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)-CO--),
ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH2-), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2-NH--), hydroxyethylene
bonds (--CH(OH)--CH2-), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH2-CO--), wherein R is the "normal"
side chain, naturally presented on the carbon atom.
[0145] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0146] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as TIC,
naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0147] In addition to the above, the peptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0148] The term "amino acid" or "amino acids" is understood to
include the 20 naturally occurring amino acids; those amino acids
often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0149] Tables 1-2 below list all the naturally occurring amino
acids (Table 3) and non-conventional or modified amino acids (Table
4).
TABLE-US-00003 TABLE 3 Three-Letter One-letter Amino Acid
Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid
Glu E Glycine Gly G Histidine His H Isoleucine Iie I Leucine Leu L
Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine
Val V Any amino acid as Xaa X above
TABLE-US-00004 TABLE 4 Non-conventional amino acid Code
Non-conventional amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.- Mgabu
L-N-methylarginine Nmarg methylbutyrate aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic Nmasp
acid aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate
L-N-methylglutamic Nmglu acid cyclohexylalanine Chexa
L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-
Nmmet methylmethionine D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu
L-N-methylornithine Nmorn D-histidine Dhis L-N- Nmphe
methylphenylalanine D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N- Nmetg
methylethylglycine D-serine Dser L-N-methyl-t- Nmtbug butylglycine
D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr .alpha.-methyl- Maib aminoisobutyrate D-valine Dval
.alpha.-methyl-.gamma.- Mgabu aminobutyrate D-.alpha.-methylalanine
Dmala .alpha.- Mchexa methylcyclohexylalanine
D-.alpha.-methylarginine Dmarg .alpha.- Mcpen
methylcyclopentylalanine D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.- Manap napthylalanine
D-.alpha.-methylaspartate Dmasp .alpha.- Mpen methylpenicillamine
D-.alpha.-methylcysteine Dmcys N-(4- Nglu aminobutyl)glycine
D-.alpha.-methylglutamine Dmgln N-(2- Naeg aminoethyl)glycine
D-.alpha.-methylhistidine Dmhis N-(3- Norn aminopropyl)glycine
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.- Nmaabu
methylbutyrate D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine
Anap D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2- Ngln carbamylethyl)glycine
D-.alpha.-methylornithine Dmorn N- Nasn (carbamylmethyl)glycine
D-.alpha.- Dmphe N-(2- Nglu methylphenylalanine
carboxyethyl)glycine D-.alpha.-methylproline Dmpro N- Nasp
(carboxymethyl)glycine D-.alpha.-methylserine Dmser
N-cyclobutylglycine Ncbut D-.alpha.-methylthreonine Dmthr
N-cycloheptylglycine Nchep D-.alpha.-methyltryptophan Dmtrp
N-cyclohexylglycine Nchex D-.alpha.-methyltyrosine Dmty
N-cyclodecylglycine Ncdec D-.alpha.-methylvaline Dmval
N-cyclododeclglycine Ncdod D-.alpha.-methylalnine Dnmala
N-cyclooctylglycine Ncoct D-.alpha.-methylarginine Dnmarg
N-cyclopropylglycine Ncpro D-.alpha.-methylasparagine Dnmasn N-
Ncund cycloundecylglycine D-.alpha.-methylasparatate Dnmasp N-(2,2-
Nbhm diphenylethyl)glycine D-.alpha.-methylcysteine Dnmcys N-(3,3-
Nbhe diphenylpropyl)glycine D-N-methylleucine Dnmleu
N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys
N-methyl-.gamma.- Nmgabu aminobutyrate N- Nmchexa D-N- Dnmmet
methylcyclohexylalanine methylmethionine D-N-methylornithine Dnmorn
N- Nmcpen methylcyclopentylalanine N-methylglycine Nala D-N- Dnmphe
methylphenylalanine N- Nmaib D-N-methylproline Dnmpro
methylaminoisobutyrate N-(1- Nile D-N-methylserine Dnmser
methylpropyl)glycine N-(2- Nile D-N-methylserine Dnmser
methylpropyl)glycine N-(2- Nleu D-N-methylthreonine Dnmthr
methylpropyl)glycine D-N-methyltryptophan Dnmtrp N-(1- Nva
methylethyl)glycine D-N-methyltyrosine Dnmtyr N-methyla- Nmanap
napthylalanine D-N-methylvaline Dnmval N- Nmpen methylpenicillamine
.gamma.-aminobutyric acid Gabu N-(p- Nhtyr hydroxyphenyl)glycine
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t- Mtbug
butylglycine L-.alpha.-methylcysteine Mcys L-methylethylglycine
Metg L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2- Nmet
methylthioethyl)glycine D-N-methylglutamine Dnmgln N-(3- Narg
guanidinopropyl)glycine D-N-methylglutamate Dnmglu N-(1- Nthr
hydroxyethyl)glycine D-N-methylhistidine Dnmhis N- Nser
(hydroxyethyl)glycine D-N-methylisoleucine Dnmile N- Nhis
(imidazolylethyl)glycine D-N-methylleucine Dnmleu N-(3- Nhtrp
indolylyethyl)glycine D-N-methyllysine Dnmlys N-methyl-.gamma.-
Nmgabu aminobutyrate N- Nmchexa D-N- Dnmmet methylcyclohexylalanine
methylmethionine D-N-methylornithine Dnmorn N- Nmcpen
methylcyclopentylalanine N-methylglycine Nala D-N- Dnmphe
methylphenylalanine N- Nmaib D-N-methylproline Dnmpro
methylaminoisobutyrate N-(1- Nile D-N-methylserine Dnmser
methylpropyl)glycine N-(2- Nleu D-N-methylthreonine Dnmthr
methylpropyl)glycine D-N-methyltryptophan Dnmtrp N-(1- Nval
methylethyl)glycine D-N-methyltyrosine Dnmtyr
N-methylanapthylalanine Nmanap D-N-methylvaline Dnmval N- Nmpen
methylpenicillamine .gamma.-aminobutyric acid Gabu N-(p- Nhtyr
hydroxyphenyl)glycine L-t-butylglycine Tbug N-(thiomethyl)glycine
Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe
L-.alpha.-methylalanine Mala L-.alpha.-methylarginine Marg
L-.alpha.-methylasparagine Masn L-.alpha.-methylaspartate Masp
L-.alpha.-methyl-t- Mtbug butylglycine L-.alpha.-methylcysteine
Mcys L-methylethylglycine Metg L-.alpha.-methylglutamine Mgln
L-.alpha.-methylglutamate Mglu L-.alpha.-methylhistidine Mhis
L-.alpha.- Mhphe methylhomophenylalanine L-.alpha.-methylisoleucine
Mile N-(2- Nmet methylthioethyl)glycine L-.alpha.-methylleucine
Mleu L-.alpha.-methyllysine Mlys L-.alpha.-methylmethionine Mmet
L-.alpha.-methylnorleucine Mnle L-.alpha.-methylnorvaline Mnva
L-.alpha.-methylornithine Morn L-.alpha.- Mphe
L-.alpha.-methylproline Mpro methylphenylalanine
L-.alpha.-methylserine mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methylvaline Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylleucine Mval L-N- Nmhphe Nnbhm
methylhomophenylalanine N-(N-(2,2-diphenylethyl) N-(N-(3,3-
diphenylpropyl) carbamylmethyl-glycine Nnbhm
carbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2- Nmbc diphenyl
ethylamino)cyclopropane
[0150] The peptides of the present invention may be utilized in a
linear form, although it will be appreciated that in cases where
cyclicization does not severely interfere with peptide
characteristics, cyclic forms of the peptide can also be
utilized.
[0151] The peptides of the present invention may be synthesized by
any techniques that are known to those skilled in the art of
peptide synthesis. For solid phase peptide synthesis, a summary of
the many techniques may be found in J. M. Stewart and J. D. Young,
Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco),
1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p.
46, Academic Press (New York), 1973. For classical solution
synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1,
Academic Press (New York), 1965.
[0152] In general, these methods comprise the sequential addition
of one or more amino acids or suitably protected amino acids to a
growing peptide chain. Normally, either the amino or carboxyl group
of the first amino acid is protected by a suitable protecting
group. The protected or derivatized amino acid can then either be
attached to an inert solid support or utilized in solution by
adding the next amino acid in the sequence having the complimentary
(amino or carboxyl) group suitably protected, under conditions
suitable for forming the amide linkage. The protecting group is
then removed from this newly added amino acid residue and the next
amino acid (suitably protected) is then added, and so forth. After
all the desired amino acids have been linked in the proper
sequence, any remaining protecting groups (and any solid support)
are removed sequentially or concurrently, to afford the final
peptide compound. By simple modification of this general procedure,
it is possible to add more than one amino acid at a time to a
growing chain, for example, by coupling (under conditions which do
not racemize chiral centers) a protected tripeptide with a properly
protected dipeptide to form, after deprotection, a pentapeptide and
so forth. Further description of peptide synthesis is disclosed in
U.S. Pat. No. 6,472,505.
[0153] One method of preparing the peptide compounds of the present
invention involves solid phase peptide synthesis. Large scale
peptide synthesis is described by Andersson Biopolymers 2000;
55(3):227-50.
[0154] Alternatively, and additionally, modifications can be
introduced into the amino acid sequence of the SP1 polypeptide by
genetic methods, by modifying the nucleic acid coding sequence
(substitutions, deletions, insertions etc) and expressing the
sequence in a transformed cell or organism, thereby producing a
modified recombinant SP1 variant polypeptide. Methods of
modification at the genetic level include, but are not limited to,
site directed mutagenesis and random mutagenesis. Signals for post
translational modification of the recombinant polypeptide, such as
glycosylation, can also be introduced into the coding sequence.
Thus, according to another aspect of the present invention, there
is provided an isolated polynucleotide comprising a nucleic acid
sequence encoding a modified SP1 polypeptide having an amino acid
sequence as set forth in any of SEQ ID NOs: 2-30). The nucleotide
sequence encoding the wild-type P. tremula SP1 polypeptide is as
set forth in SEQ ID NO: 149.
[0155] It will be appreciated that the polynucleotide of the
present invention can be introduced into a vector for recombinant
expression in a host organism. According to another aspect of the
present invention there is provided a nucleic acid construct
comprising the isolated nucleic acid described herein.
[0156] According to a preferred embodiment the nucleic acid
construct according to this aspect of the present invention further
comprising a promoter for regulating the expression of the
polynucleotide in a sense orientation. Such promoters are known to
be cis-acting sequence elements required for transcription as they
serve to bind DNA dependent RNA polymerase which transcribes
sequences present downstream thereof.
[0157] While the polynucleotide described herein is an essential
element of the invention, it can be used in different contexts. The
promoter of choice that is used in conjunction with the
polynucleotide of the invention is of secondary importance, and
will comprise any suitable promoter. It will be appreciated by one
skilled in the art, however, that it is necessary to make sure that
the transcription start site(s) will be located upstream of an open
reading frame. In a preferred embodiment of the present invention,
the promoter that is selected comprises an element that is active
in the particular host cells of interest, be it a bacteria, yeast
or a higher cell of a plant or animal.
[0158] A construct according to the present invention preferably
further includes an appropriate selectable marker. In a more
preferred embodiment according to the present invention the
construct further includes an origin of replication. In another
most preferred embodiment according to the present invention the
construct is a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells, or integration in the genome, of an organism of choice. The
construct according to this aspect of the present invention can be,
for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a
virus or an artificial chromosome.
[0159] The construct of the present invention can be used to
express the polypeptide encoded thereby in a variety of species
ranging from bacteria such as E. coli, yeast cells or higher cells
such as the cells of a plant. Expression can be selected stable or
transient.
[0160] For effecting plant transformation, the exogenous
polynucleotides which encode enzymes capable of catalyzing proline
production are preferably included within a nucleic acid construct
or constructs which serve to facilitate the introduction of the
exogenous polynucleotides into plant cells or tissues and the
expression of the enzymes in the plant.
[0161] Since the polypeptides of the present invention retain their
SP-1 activities (as mentioned hereinabove) they can be used in a
myriad of applications such as previously described, and currently
envisaged, as further described hereinbelow. It will be appreciated
that where desirable, native SP-1 polypeptides may also be used in
accordance with the present invention.
[0162] According to one aspect of the present invention, there is
provided a method of delivering a therapeutic, diagnostic or
cosmetic agent to a subject in need thereof, wherein the method
comprises administering to the subject a therapeutically,
cosmetically or diagnostically effective amount of a composition of
matter comprising an SP1-polypeptide of the present invention in
molecular association with the agent. In a preferred embodiment,
the SP 1 polypeptide is a modified SP1 polypeptide. In another
embodiment, the molecular association with the agent is a
reversible association.
[0163] As used herein, the phrase "therapeutic agent" refers to any
agent, the administration of which is capable of causing an
improvement in any aspect of a given condition. A therapeutic agent
may be symptomatically effective, partially effective, may cure,
treat, palliate, prevent the progression of, improve the prognosis
for, etc any condition for which it is administered. Therapeutic
agents can be effective alone, or as adjuncts to other agents.
Therapeutic agents can be effective in short and/or long term, and
can be broadly effective within a wide range of conditions, or
narrow and specific in their effectiveness.
[0164] As used herein, the phrase "diagnostic agent" refers to any
agent which is used in connection with methods for diagnosing the
presence or absence of a disease or condition in a patient.
Exemplary diagnostic agents include, for example, contrast agents
for use in connection with ultrasound, magnetic resonance imaging
or computed tomography of a patient.
[0165] As used herein, the term "cosmetic agent" refers to any
agent, such as a pigment or fragrance, which may be topically
applied to human skin for aesthetic effect and which preferably
does not cause irritation. Cosmetic agents are well known in the
art and are included in such products as lipsticks, eye shadows,
rouges, foundations and other forms of "make-up", creams, pastes,
lotions, balms, sprays, gels, foams, etc. that can be applied
dermally or topically, such as creams, e.g., grease creams or dry
creams.
[0166] As used herein, the phrase "subject in need thereof" refers
to any subject which may derive benefit from the administration of
the composition-of-matter of the present invention. Such a subject
can be, for example, a subject having a specific condition, or at
risk of having a specific condition, for which the administration
of the composition of matter can have a therapeutic or beneficial
effect.
[0167] The composition of matter of the present invention can be
administered to an organism per se, or in a pharmaceutical
composition where it is mixed with suitable carriers or
excipients.
[0168] The composition-of-matter of the present invention can be a
pharmaceutical composition. As used herein a "pharmaceutical
composition" refers to a preparation of one or more of the active
ingredients described herein with other chemical components such as
physiologically suitable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration of a
compound to an organism.
[0169] Herein the term "active ingredient" refers to the SP1 or SP1
variant, alone or in molecular association with a substance or
agent, accountable for the biological effect.
[0170] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0171] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0172] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0173] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, inrtaperitoneal, intranasal, or
intraocular injections.
[0174] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0175] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0176] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0177] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0178] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0179] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0180] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0181] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0182] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0183] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0184] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0185] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0186] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0187] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients (nucleic acid
construct) effective to prevent, alleviate or ameliorate symptoms
of a disorder (e.g., ischemia) or prolong the survival of the
subject being treated.
[0188] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0189] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0190] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0191] Dosage amount and interval may be adjusted individually to
provide plasma or brain levels of the active ingredient are
sufficient to induce or suppress the biological effect (minimal
effective concentration, MEC). The MEC will vary for each
preparation, but can be estimated from in vitro data. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. Detection assays can
be used to determine plasma concentrations.
[0192] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0193] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0194] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as if further detailed
above.
[0195] While reducing the present invention to practice, it was
uncovered that the introduction of disulfide bridges into SP1 amino
acid sequence produces a variant SP1 polypeptide having unique
properties. N-terminal cysteine residues (Cys 2 P1 variant, SEQ ID
NO: 3) sensitizes the variant SP1 polypeptide to the redox state
(FIG. 8b, Example 4). Thus, modified SP1 offers specific control
over complexing and release (FIGS. 8, 9, 10 and 11, Example 4) of
agents or substances, in this case the labeling substance FA.
Indeed, release of drug molecules from SP1-drug complexes such as
Cys 2 SP1-DOX, and fluorescent label from Cys 2 SP1-FA complexes
was shown to be sensitive to redox state (FIGS. 9-11, 14, 16,
Example 4). It will be appreciated that the previously unattainable
control of complex formation can be advantageous in designing
modified SP1 polypeptide drug carriers, diagnostic tools,
nanostructures, etc. For example, SP1-complexes with substances
such as therapeutic agents, cosmetic agents, fragrance agents,
diagnostic agents, etc. can be induced to formation by exposure to
reducing conditions, and at a later point, induced to release by
competition for the thiol bearing residues (see schematic, FIG.
8a-c).
[0196] While reducing the present invention to practice, SP1
modified with specific tumor vasculature recognition peptides
showed characteristic oligomer complex formation, heat stability
and protease resistance (FIG. 4, Ex 2). Thus, SP1 can be modified
to comprise a target recognition sequence or moiety, and further
modified to form a reversible complex with a substance
(therapeutic, diagnostic, conductive agent, etc). Thus, such a
modified SP1 polypeptide can be used to target the delivery of a
substance in reversible complex with SP1.
[0197] It will be appreciated that complexing of cytotoxic drug to
nanoparticles such as the SP1 oligomer may also assist passive
targeting to tumors. Several model tumour systems are now known to
display increased vascular permeability compared with normal
tissues, permitting their selective targeting using macromolecular
drug carriers. Preliminary clinical observations suggest that
increased vascular permeability is characteristic of some types of
human cancer, and this may have important implications for the use
of carriers such as SP1 in facilitating macromolecular drug
treatments, including cytotoxic drugs, antibody targeting and
delivery of DNA for gene therapy.
[0198] The modified SP1 polypeptides of the present invention can
be used to stabilize a substance. As used herein, the term
"stabilize" refers to increasing the chemical, physical or
biochemical stability of a molecule, composition, compound, etc.
Stability can be further defined in the context of specific
properties. In a preferred embodiment, stability is temperature
stability, ionic strength stability, protease stability and
catalytic stability. Examples of assays for measuring such
stability are described in detail hereinbelow.
[0199] While reducing the present invention to practice, it was
shown that complexing of molecules with SP1 greatly enhanced
solubility and stability in solution. As used herein, the term
"solubility" refers to the ability of a solute to be evenly
dispersed and dissolved in a solvent, in order to form a solution
comprising the solvent and solute. It will be appreciated that all
solutes are, in theory, soluble in all solvents. However, poorly or
negligibly soluble (immiscible) solutes do not form solutions of
any significant concentration with given solvents.
[0200] Thus, as used herein, "enhancing the solubility of a
substance in a solution" refers to increasing the concentration of
said substance, as a solute, in a solution with a solvent. In a
preferred embodiment, the substance is a hydrophobic substance,
typically insoluble or poorly soluble in water, and the solvent is
an aqueous solvent.
[0201] The stability of unmodified, and modified SP1 polypeptide
oligomeric complex to organic solvents was shown in FIG. 18
hereinbelow. When combined with hydrophobic molecules such as PTX
in organic solvents, dried and reconstituted in aqueous solvent,
molecular association and complex formation between the SP1 and the
hydrophobic molecule rendered the PTX water soluble (FIGS. 19 and
20). Thus, SP1 can be used to enhance the solubility of a substance
in a solution. In a preferred embodiment, the substance is a
hydrophobic substance, and the solution is an aqueous solution.
Such substances, such as hydrophobic drugs, volatile esters and
other molecules, fragrance molecules, cosmetic molecules, oils,
pigments, vitamins, organic molecules, etc. can be solubilized for
mixing with water and aqueous solvents. Further, while reducing the
present invention to practice, it was shown that the lyophilized
SP-PTX complex remains stable after reconstitution (see Example 5
hereinbelow). Thus, molecular association and complex formation
with SP1 and modified SP1 variants can be used for more effective
storage of hydrophobic, volatile, labile, etc. molecules such as
PTX.
[0202] As mentioned hereinabove, the modified SP1 polypeptides of
the present invention can bear more than one modification in the
amino acid sequence. Thus, for example, SP1 polypeptides modified
capable of complexing with inorganic molecules (e.g. metal ions;
see Table 1 hereinabove) can be further modified to form a
reversible molecular association with a second substance, and used
to deliver the second substance to any surface comprising the
inorganic molecule. Such an association can be useful in
nanotechnology and solid-state engineering applications, wherein
the modified SP1 complex can be used to deposit a layer of the
second substance, evenly and of a predetermined molecular
thickness. Such a method of delivery of, for example, doping or
insulating substances, would be particularly advantageous for use
over uneven surfaces.
[0203] The modified SP1 polypeptides of the present invention can
be used for controlled delivery and release of inorganic molecules
in biomimetic applications. SP1 has been shown to self assemble in
ordered geometric conformations (see FIG. 1 and FIG. 2), which can
be altered by modification of the amino acid sequence. Further
modifications to provide carrier capability (such as reversible
binding of inorganic molecules) can produce SP1 molecules modified
to serve as molecular carriers in biomimetic processes, such as
controlled crystal formation, etc. Such modified SP1 polypeptides
can be useful for accurate delivery and release of, for example,
inorganic molecules, to prevent uncontrolled aggregation in
nanoscale processes, and as coupling molecules having well-defined
and controllable properties (see Sarikaya et al., Ann Rev Mater Res
2004; 34:373-408 for a recent review of the subject of
biomemtics).
[0204] The SP1 polypeptides of the present invention can be used to
make and operate nanoscale structures and devices. Polypeptides
have an advantage in nanoscale technology, since binding peptides
and proteins are selected and designed at the molecular level and
through genetics, allowing control at the lowest dimensional scale
possible. Also, such proteins can be used as linkers or "molecular
erector sets" to join synthetic entities, including nanoparticles,
functional polymers, or other nanostructures on molecular
templates. Further, biological molecules self- and coassemble into
ordered nanostructures, ensuring a robust assembly process for the
construction of complex nanostructures, and possibly hierarchical
structures, similar to those found in nature.
[0205] As mentioned hereinabove, the SP1 polypeptides of the
present invention can self-assemble to form regular and predictable
nano-scale structures. Thus, there is provided a
composition-of-matter comprising a plurality of self assembled
modified SP1 monomers useful as, for example, molecular linkers and
complex nanosructures.
[0206] The SP1 polypeptides of the present invention can further be
used for affinity binding (through specific fused peptides or
polypeptides) of ligand and ligand binding molecules, surface
coating of any compounds and/or molecules capable of molecular
association and complexing with SP1 oligomers, nanocircuitry
through controlled association of conducting molecules or
semiconductors in molecular association with SP1 oligomers,
magnetic particles or nanoparticles associated with SP1 oligomers,
controlled association and release of any biologically active
molecules, such as herbicides, insecticides, volatile and
odoriferous compounds, etc., nano-computing, lithography and
printing with conductive inks, nanoarchitecture through controlled
association and dissociation of three dimensional nanostructures.
Yet further, the SP1 polypeptides of the present invention can be
incorporate as a component of a conductive device such as an
electronic device.
[0207] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0208] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0209] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Materials and Experimental Methods
[0210] Expression of recombinant SP1
[0211] A 567-bp cDNA clone was isolated by screening
7.sub.--10.sup.5 recombinant phage plaques from a lambda expression
library derived from water-stressed aspen shoots, using anti-SP1
antibodies (Wang et al, U.S. patent application Ser. No.
10/233,409). E. coli strain BL21(DE3) was transformed with a
plasmid carrying the sp1 gene (pET29a, kanamycin resistance
conferred) (Wang, et al. Plant Phys 2002; 130:865-75). Wild type
SP1 as well as its variants were generated and expressed in E.
coli: a full length SP1 without any additional tag, designated as
SP1 (SEQ ID NO:1); a six-histidine tag was introduced to the
N-terminal of SP1 to generate 6HSP1 (SEQ ID NO:7), and a cysteine
residue was introduced in position 21 of SP1 to generate Cys2 SP1
(SEQ ID NO:3), and the .DELTA.NSP1 (SEQ ID NO:2) was generated by a
deletion of amino acid 2-6 at SP1 N-terminus. The expression of
these recombinant SP1 proteins followed standard recombinant
procedures, as described in Wang et al., (Acta Crys 2003;
D59:512-14).
[0212] Protein Purification
[0213] Recombinant SP1 was produced from E. coli as described in
Wang et al 2003. 6HSP1 was further purified on Ni-NTA Agarose beads
(P-6611, Sigma Chemicals St Louis Mich.) according to the
supplier's protocol except for the fact that the elution buffer
contained 400 mM imidazole. The boiling stable fraction of
.DELTA.NSP1 was dialyzed against 2.times.20 volumes of 15-20 mM
piperazine at pH 5.9 as preparation to the anion exchange column
SOURCE-15Q (Amersham Biosciences UK). Buffer A in the mobile phase
was 20 mM piperazine pH 5.1 and the same buffer with 1M NaCl was
used as buffer B. The .DELTA.NSP1 was eluted by 23-25% buffer B.
Ammonium sulfate at a final concentration of 1M and NaOH to a 7.5
pH were added to the purified .DELTA.NSP1, and then loaded to a
HiTrap phenyl sepharose HP column (Amersham Biosciences UK) that
was prewashed with 50 mM phosphate buffer containing 1M Ammonium
Sulfate at pH 7.5. The .DELTA.NSP1 eluted at 47-48% 50 mM phosphate
pH 7.5 (buffer B). The .DELTA.NSP1 was then concentrated and
diafiltered by 30 kDa cut-off ultra filtration concentrator using
25 mM phosphate pH 7.5).
[0214] Analytical Ultracentrifugation
[0215] Equilibrium sedimentation studies were carried out using a
Beckman Optima.TM. XL-1 analytical ultracentrifuge (Beckman
Instruments, INC.). Aspen SP1 was dialysed overnight against
200-fold 20 mM Tris-HCl, pH 8.0. The samples were then diluted with
dialysate to generate protein solutions of approximately 202, 152,
68, 22.5 and 6.5 .mu.M. The samples were spun in a six-sector cell
at rotor speeds of 6000 and 7000 rpm at 20.degree. C. Data were
collected at 280, 220 and 254 nm and were analyzed using the
following equation:
M=[2RT/(1-.sigma.).rho..omega..sup.2][d(ln(c))/dr.sup.2)] with a
typical .nu.=0.73 cm.sup.3 g.sup.-1 and .rho.=0.9994 g
cm.sup.-1.
[0216] Transmission Electron Microscopy (TEM) Study
[0217] SP1 (0.05 mg/ml) was applied to glow-discharged, carbon and
nitrocellulose-coated copper 400-mesh grids, and stained with 2%
uranyl acetate. Images were taken with an FEI Tecnai-12 microscope
and recorded on Kodak S0163 film or on a Megaview III digital
camera (Soft Imaging Systems, Munster, Germany). Micrographs were
digitized with an Imacon Flextight II scanner. Image processing for
averaging of top-view wild-type SP1 particles was done with the
SPIDER program suite (Frank et al., 1996) and consisted of an
initial reference-free alignment (translational and rotational)
followed by three rounds of reference-based alignment.
Cryo-negative stained images of SP1 were prepared by placing 4
.mu.l of SP1 (1 mg/ml) on lacey grids (SPI Supplies, West Chester
Pa.), applying 16% ammonium molybdate (Adrien et al. 1998), and
plunging in liquid ethane. Imaging under low dose cryo-conditions
was done on an FEI Tecnai F20 microscope and recorded on a TVIPS
(Gauting, Germany) 1 k.times.1 k Biocam camera.
[0218] Chemical Cross-Linking of SP1 and Mass Spectrometry
[0219] SP1 at 1 mg/ml was incubated at room temperature with 0.25%
gluteraldehyde (GA) in 50 mM Triethanolamine buffer (pH 5.7, 72
hr). The non-cross-linked and cross-linked SP1 products were
subjected to mass spectrometry analysis. Matrix-assisted
laser-desorption time-of-flight mass-spectrometry (MALDI-TOF-MS)
was performed on a Micromass TofSpec 2E reflectron
mass-spectrometer (The Protein Research Center, Technion, Haifa,
Israel).
[0220] SP1 Stability Following Exposure to SDS and Heating
[0221] SP1 (20 .mu.g) was prepared in SDS sample buffer at
different SP1-monomer: SDS molar ratios and boiled (or not) (5 min)
prior to SDS-PAGE analysis. Heat stability of SP1 oligomer (10
.mu.g) was tested in the same buffer at SP1-monomer: SDS=1:1733 and
heated for 1 to 10 min at different temperatures.
[0222] Protease Susceptibility Examination of SP1-SP1
[0223] (10 .mu.g) prepared either in V8 protease digestion buffer
(125 mM Tris-HCl, pH 6.8, 10% glycerol, 0.5% SDS) or standard
buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl), was boiled
for 2 mM or not boiled prior the addition of protease
(Staphylococcus aureus V8 protease, trypsin, proteinase K, Sigma
Chemicals Inc., Israel). Proteases were added to a final
concentration of 50 .mu.g/ml (SP1: protease equal to 1:20, w/w).
The digestion was performed at 37.degree. C. for 1 hr. The samples
were then prepared in SDS sample buffer, and boiled or not before
being subjected to 17% tricine-SDS-PAGE.
[0224] Purification of SP1 by Boiling and Proteolysis
[0225] Plant total soluble proteins or concentrated total
boiling-soluble proteins were incubated with 50 .mu.g/ml proteinase
K (37.degree. C., 1 hr). Similar procedures were applied to total
recombinant bacterial proteins. PK was inactivated by boiling (10
min) followed by centrifugation (15,000.times.g, 10 min). SP1 was
concentrated by ultrafiltration (10 kDa cut-off, VIVASCIENCE,
Binbrook Lincoln, England).
[0226] Engineering Disulfide Bridges to SP1 Complex.
[0227] Cys 2SP1 gene was constructed by site directed mutagenesis
of the N terminal alanine to cysteine. Both wild type and Cys 2 SP1
(SEQ ID NO:3) (2 mg/ml) were incubated in the presence or absence
of 10 mM DTT overnight and preboiled with a 2% SDS sample buffer
prior to SDS-PAGE analysis.
[0228] Resistance of SP1 Complex to Various Organic Solvents.
[0229] 150 ul of 1 mg/ml samples in 10 mM sodium phosphate pH 7
were lyophilized overnight and resuspended in 150 .mu.l organic
solvents for 10 minutes. Following a 30 minutes speed vac the
samples were resuspended in 150 .mu.l water and analyzed by
SDS-PAGE.
[0230] Reassembly of SP1 Hetero-Oligomers.
[0231] Purified 6HSP1 and .DELTA.NSP1 were first denatured to the
monomeric forms by boiling the proteins in SDS sample buffer. The
denatured proteins were then separated in preparative SDS-PAGE, and
visualized by Coomassie blue staining. The monomeric forms of two
recombinant protein bands were excised. Gel slices that carried
monomeric form of 6HSP1 (SEQ ID NO:7) and .DELTA.NSP1 (SEQ ID NO:2)
were mixed at 1:1 ratio (v/v). In order to enhance the
surface/volume ratio, the gel slices were pulverized using a pestle
and mortar in the presence of liquid nitrogen. Then the proteins
were co-eluted by electro-elution as described previously (Wang et
al., 2002). The hetero-oligomeric complex was isolated by
subjecting the eluated protein to Ni-NTA Agarose beads and the
bound proteins were eluted by 400 mM imidazole using a standard
procedure (Sigma protocol for P 6611). Proteinase K which digests
monomeric SP1 but not SP1 complex (described in this paper), was
employed to eliminate the monomeric form of 6HSP1 (SEQ ID NO:7) and
.DELTA.NSP1 (SEQ ID NO:2) from the Ni-NTA purified proteins. The
composition of hetero-oligomeric SP1 was determined by SDS-PAGE and
visualized by silver staining.
[0232] Ultrafiltration:
[0233] Ultrafiltration was used for the detection of complex
formation with the water soluble ligands FA and DOX which have a
distinctive absorption properties and can be determined by
spectroscopic measurements. Free FA and DOX are much smaller than
SP1 (0.35, and 0.58 versus 150 kDa, respectively): while the free
FA and DOX pass through a molecular weight cutoff membrane of 30
kDa (flow through-fraction), both free SP1 and SP1 complexes are
retained above the membrane (retained fraction). Several additional
washing cycles remove all remaining free FA and DOX, and the
ligand-SP1 complex remains in the retained fraction.
[0234] Size Exclusion Chromatography:
[0235] Size exclusion chromatography is a common method for
separation of molecules of different sizes under mild conditions
and was employed to test FA and DOX complex formation under mild
conditions. SP1 is eluted from the column after 7 min and is
detected at 278 nm only, and free FA is eluted from the column (TSK
G3000 SWXL, Tosohaas) after 16 mM and is detected at 490 nm. The
SP1-FA and SP1-DOX complexes also eluted at the same time but is
detected also at 490 and 475 nm, respectively. SP1 is eluted from
the column after 7 min and is detected at 278 nm only. DOX/SP1
ratio is determine from the standard curve obtained in
solution.
[0236] C-18 Reversed Phase HPLC:
[0237] Reverse phase HPLC (RP-HPLC) analysis separates between free
DOX, PXT, and SP1. Both compounds bind to the resin (C-18) and are
eluted at different acetonitrile concentrations, and detected at
both 278 and 225 nm (SP1), 225 nm (PTX) and 477 nm (DOX).
[0238] SP1-DOX complexes together with uncomplexed SP1 and are
detected at 477 nm, as well as 278 nm. Quantification of SP1-DOX,
and free DOX is directly calculated from the absorbance in their
peaks at 477 nm. However to estimate the amount of protein in the
SP1-DOX peak, absorption at 278 nm is corrected for DOX according
to the following equation (OD278-0.77*OD.sub.477). In contrast with
FA and DOX, complexed PTX cannot be detected, but it is detected in
the same elution as free PTX.
[0239] The C18 RP HPLC separation, and detection of DOX and PTX
compounds is outlined in below. Solvent A=water+0.1% TFA. Solvent
B=Acetonitrile+0.1% TFA. Program was 0-5 min 75% A, 0% B; 5-15 min
25%-75% B. SP1 was detected at 225 and/or 278 nm; DOX at 477 and/or
278 nm; and PTX at 225 nm.
[0240] DOX-SP1 Complex:
[0241] Pyrogen free Cys2 SP1 variant (SEQ ID NO:3) in 20 mM (Na
Phosphate buffer pH-6.7) final 2 mg/ml was diluted with DOX
solution (Teva, Israel) 1:2 dilution final 1 mg/ml. Where
indicated, GSH was added, and the solution is mixed overnight.
Where indicated (oxidized), H.sub.2O.sub.2 is added to 0.1%, buffer
added to 3.times. volume, and the solution is sonicated (3.times.22
sec. 1:1 pulse/pause at 3% amplitude 0.5 min. pause, using a
Vibra-Cell 750W sonicator).
[0242] Ethanol Precipitation of Free DOX:
[0243] Solution diluted 5.times. (V:V) with ethanol, incubated
-20.degree. C. for 3 hours, centrifuged 30 minutes at 1250.times.g,
room temperature. Pelleted material is washed and resuspended in
cold ethanol, repelleted, resuspended and analyzed by HPLC.
[0244] Removal of Unbound DOX:
[0245] The SP1-DOX solution was washed by ultrafiltration 30K
cut-off microcon filter (Millipor Ltd., Billerica, Mass.), then
washed with Na Phosphate buffer (pH 6.7), and PBS, until
flow-through is colorless.
[0246] SP1-PTX Complex:
[0247] 3 mg SP1 (25 mg/ml, 120 ul in PBS) was freeze dried for 6 h
in 15 ml plastic tubes. 300 ul PTX (1 mg/ml in dry aceton/hexan
1:1+0.1% betamercaptoethanol) was added, and aceton/hexan+0.1%
betamercaptoethanol added to a final volume of 4 ml. The mixture
was sonicated (1 sec pulse, 3 second pause, 45'' sonication time,
total 3 min, 35% intensity, on ice). The organic solvents were
evaporated by dessication overnight, 0.4 ml PBS added, the mixture
sonicated (1 sec pulse, 3 sec pause, 30'' sanitation time, total
2.5 min, 35% intensity, on ice). Debris was pelleted by
centrifugation (5 min, 14000 RPM). For tissue culture experiments,
aliquots were filtered.
[0248] Cell Growth Conditions:
[0249] Human colon adenocarcinoma (HT-29) cells. The cells were
grown in 50-ml flasks containing DMEM medium (Biological
Industries, Bet Haemek), supplemented with 10% fetal calf serum, 1%
glutamine and 1% Antibiotic-Antimicotic solution (Biolab, Israel).
The cells were trypsinizated, and 2 ml medium containing
5.times.10.sup.4 cells were plated in each well of a 6-wells plate.
SP1, drugs or SP1-complexed with drugs was added at the indicated
concentrations. The cells were incubated at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2. After 48 hours the
medium in the presence or absence of drugs was replaced in each
well respectively, to maintain a constant supply of ingredients and
drugs. After four days the medium was removed and the number of
viable cells in the cultures was determined by spectroscopy.
[0250] In Vivo Effects of SP1-DOX and SP1-PTX in B16 Melanoma
Model;
[0251] B16-F10 (B16) melanoma tumor C57Bl male mice bearing the
B16-F10 (B 16) melanoma tumor were prepared and cared for as
described by Kalechman (Int J Cancer 2000; 86:281-8). B16F10
melanoma cells (5.times.10.sup.5 or 5.times.10.sup.6 cells/mouse)
were injected into the lateral tail vein of the mice and the
presence of cancer tumors was evaluated at day 14 following
melanoma cell injection by an overall view of the mouse and/or
histopathology examinations. Mice were divided into three groups:
Group A--injected once (iv with Fluoresceinamine-SP1 complex
solution (SP1-FA conjugate, 10 mg/ml in PBS; 0.1 ml per animal).
N=5 mice. Group B received FA once, at 1 week. N=5 mice. Group C
received no injections N=2 mice. 24 h post injection internal
organs were collected and stored at -70.degree. C. Blood was
collected, coagulated at room temperature, sera separated and
frozen.
[0252] Organs and tumors were homogenized, and extracts and diluted
(3.times. in PBS) sera were heat treated (85.degree. C. for 30
minutes), and separated on SDS PAGE. For immunodetection, separated
proteins were transferred from gel to nitrocellulose paper by
electroblotting. Nitrocellulose blots was blocked by immersion in
Tris-buffered saline+0.05% Tween 20, pH=7.7 (TBST) containing 3%
skimmed milk. After washing the skimmed milk with TBST, the
nitrocellulose blot was immersed in primary rabbit anti
SP1-antibody in TBST. After washing primary antibody with TBST, the
nitrocellulose blot was immersed in secondary Goat anti-Rabbit
antibody HRP conjugate in TBST. After washing excess secondary
antibody with TBST, the nitrocellulose blot was contacted with the
HRP chemiluminescent substrate (ECL).
[0253] Photographic film is exposed to the wrapped nitrocellulose
paper, then developed and fixed.
[0254] In Vivo Effects of Free DOX and SP1-DOX on Tumor Size:
[0255] Human LS 147T colon cancer (one million cells per animal)
were grafted sub cutaneously to CD1 nude mice, (3-4 weeks old,
18-20 g) (Meyer et al. 1995 Am J Dermatopath; 17:368-73). 8 days
later a 3-10-mm tumor appeared in the point of injection. At this
time the animals were divided into two groups (6 animals in each),
average tumor size and animal weight were similar.
[0256] 8 days after tumor grafting SP1-DOX (50 mg/Kg in PBS, about
1 mg DOX equivalent/Kg), free DOX (3 mg/Kg in PBS) or PBS alone,
six mice in each group, were injected iv to the tail vein twice a
week for four weeks. Tumor dimension was determined by caliper
measurements by the standard equation (Kalechman et al. Int J
Cancer 2000; 86:281-8). At 35 days post tumor grafting the animal
were sacrificed. Tumors were removed and their weight was
determined.
Experimental Results
Example 1
Structure of the SP1 Protein
[0257] SDS gel electrophoresis analysis of both native SP1 and its
recombinant form shows that SP1 appears in two forms: a monomer
(12.4-kDa) which appears when the sample is boiled in the gel
application buffer in the presence of SDS and in an oligometric
form (116-kDa protein) which appear when SP1 is not boiled prior to
application on PAGE (see Wang et al 2002, Dgany 2004, Wang et al
2006, U.S. patent application Ser. No. 10/443,209). Several methods
have been employed to demonstrate that SP1 in solution forms a
dodecamer with a molecular weight of 150 kDa. Equilibrium
analytical ultracentrifugation was employed to analyze the SP1
oligomeric state. As SP1 concentration approached zero, the
measured molecular mass of the SP1 particles in solution (144 kDa
at 5.6 .mu.M monomer concentration) approached the value calculated
for a dodecamer (148 kDa).
[0258] SP1 was subjected to MALDI-TOF-MS. The data revealed 12
protein peaks, of which the first (12338 Da) was close to the
predicted molecular weight of the monomer (12369 Da). The other
peaks corresponded to SP1 dimer up to a dodecamer with a molecular
interval of about 12.4 kDa. MALDI-TOF-MS analysis of cross-linked
SP1 revealed 12 clear peaks with molecular mass ranging from 12998
to 154706 Da, corresponding to the monomer, and up to a dodecamer.
Gel filtration HPLC analysis using TSK3000 column also shows that
SP1 forms a dodecamer. The oligomeric form was further estimated on
an electro-eluted high-molecular mass SP1 (116 kD) appeared as a
single peak at about 9.8 min. This peak, as calculated from a
standard curve, corresponded to a molecular mass of 144.9.+-.1.54
kD, which is 11.7 (about 12 units) of SP1 monomer (12.369 kD).
[0259] While reducing the present invention to practice, electron
microscope study of SP1 was undertaken. Electron microscopy studies
showed that SP1 is a ring-like protein with a central cavity.
[0260] In order to determine conditions under which SP1 forms two
dimensional crystals, SP1 was mixed with phospholipids (DOTAP/DOPC
1:1, w/w, in Hexane Chloroform). FIG. 1 shows a TEM image of
phospholipid induced two dimensional SP1 crystal monolayer,
indicating the ability to form two dimensional crystals of SP1.
Thus, indicating that that the particles can arrange in different
ways and SP1 can self assembled into higher order structures.
[0261] SP1 Monomer:
[0262] X-ray crystallography studies (see Dgany et al 2004,) showed
that SP1 chain has .alpha.- and .beta.-folding with three
.alpha.-helices, H1 (residues 23-39), H2a (residues 74-81), and H2b
(residues 84-93), and a .beta.-sheet formed by four antiparallel
.beta.-strands, B3 (residues 9-17), B1 (residues 45-50), B2
(residues 65-71), and B4 (residues 97-108). The N-terminal segment
points toward the solvent and is mobile as evidenced by the lack of
interpretable electron density for the first two residues and the
large temperature factors for Thr-3 and Lys-4. The long loop formed
by residues 51-64 is largely unstructured. This loop projects away
from the molecule and is involved in dimer contacts. Helices H1 and
H2 define an external convex surface with numerous hydrophilic and
acidic side chains facing toward the solvent.
[0263] The inner side of this surface and the opposing .beta.-sheet
enclose a hydrophobic central cavity rich in aromatic and
hydrophobic residues. Most of the phenylalanines in the SP1
molecule occupy this cavity (Phe-17, -46, -67, -71, -89, and -93).
Trp-48 and Tyr-33, Tyr-63 and Tyr-80, together with the two
histidines (His-11 and His-65), and Arg-100 block access of the
solvent to the cavity. Without wishing to be limited to a single
hypothesis, we believe that this cavity may serve as binding site
for small hydrophobic molecules.
[0264] It should be noted that SP1 structure is similar to the
structure of its Arabidopsis thaliana analog (gene locus
At3g1721050, accession no: AY064673, SEQ ID NO: 150)) as resolved
by both X-ray crystallography and NMR (Bingman et al. Proteins
2004; 57:218-20; Lytle et al. J Biomol NMR; 28:397-400).
[0265] Purification of SP1 protein is enabled by virtue of its
exceptional stability, which allows partial extraction by heat
treatment (see Methods, hereinabove). The resultant heat resistant
fraction is 30% pure (FIG. 6, lanes 1 and 2). Further purification
yields a chromatographically pure preparation of SP1, which can be
detected as a single peak on reverse phase HPLC and size exclusion
chromatography (FIG. 6, lane 3, FIG. 7 and FIG. 30).
[0266] SP1 Dodecamer:
[0267] The dimer-dimer contacts predominantly involve hydrophilic
side chains and charged groups or are mediated by water molecules.
These contacts take place mainly along the B1, H1, and the
N-terminal tails (see Dgany, 2004). As a result of the interdimeric
interactions six dimers create a ring-like structure around a
pseudo 6-fold axis. The ring-like structure of the dodecamers has
an outer radius of .about.50 .ANG. and an inner radius of .about.15
.ANG.. The loop including residues 18-22 in each dimer protrudes
toward the solvent, whereas the arms of the N-terminal are
extending toward the inner part of the ring-like structure. The
6-fold symmetry is broken, because the contacts between equivalent
molecules in neighboring dimers are not identical (Dgany 2004).
Example 2
SP1 is a Boiling- and Denaturing-Stable, and Protease-Resistant
Molecule
[0268] The stability of the SP1 complex in the presence of SDS was
examined by incubating purified SP1 with SDS at different molar
ratios and at different temperatures. Dissociation of the SP1
complex to monomers required a molar ratio greater than 600:1
(SDS:SP1-monomer) accompanied by boiling before loading onto the
gel. Without boiling, even at a ratio of 3467 to 1, SP1 remained as
a complex on SDS-PAGE. Incubation of SP1 with 1734-fold SDS (1%) at
temperatures of 80.degree. C. or lower did not cause significant
disassociation of the complex (Wang 2006).
[0269] SP1 protein exhibits exceptional heat stability, which
allows partial extraction of crude cellular preparations by heat
treatment, as mentioned in Example 1 hereinabove. FIG. 6 shows the
degree of purity achieved by heat treatment.
[0270] Differential scanning calorimetry study of SP1 indicates in
a Tm of 107.degree. C. for SP1. These results further support our
previous findings that SP1 is a boiling stable protein and that the
high oligomeric form dissociates only upon boiling in the presence
of 2% SDS (Dgany 2004). Further, folding and refolding of inclusion
bodies including insoluble SP1, and the heat stability of the
unfolded proteins indicate that the intact monomer, as well as the
oligomer, is heat resistant (FIG. 5).
[0271] SP1 is vulnerable to V8 protease or subtilysine (alcalase,
Novo Industries) digestion when disassociated to its monomeric form
(boiled in 0.5% SDS, or dissolved inclusion bodies). However, V8
protease was unable to digest the intact oligomer (see FIG. 5).
When the oligomer-protease mixture was further boiled in SDS sample
buffer, only the SP1 monomer was observed and no peptide fragment
was detected on the gel. When the same mixture was subjected to
SDS-PAGE analysis without boiling, an intact complex was observed.
Similar results were obtained with trypsin and proteinase K and
subtilysine, indicating the superior resistance of the SP1 complex
to a wide variety of proteases (Wang 2002, Wang 2006). Thus,
whereas properly folded SP1 protein is protease resistance,
unfolded protein, susceptible to protease, can be removed by
protease and heat treatment (FIG. 5).
[0272] In order to further test the stability of SP1 and SP1
oligomeric complexes, wild type SP1 and the SP1 variant Cys2 SP1
were dissolved in buffer, exposed to organic solvents methanol and
hexane, and analyzed by SDS-PAGE (FIG. 18). Predominantly high
molecular weight oligomer complex form was detected in all samples
treated (FIG. 18, lanes 1-3), indicating that the SP1 complex is
resistant to denaturation by organic solvents.
[0273] Thus, the SP1 complex shows surprisingly strong resistance
to temperature and detergent denaturation, organic solvents and to
proteolytic degradation.
Example 3
SP1 Variants
[0274] SP1 variants can be constructed to enhance, or otherwise
alter SP1 stability, capabilities for oligomerization and/or
binding and/or complexing with other molecules and/or ability to
form inter-subunit disulfide bonds and/or change the dimension of
the central cavity. Numerous SP1 variant proteins were constructed
to investigate the effects of specific alterations on properties of
SP1.
[0275] The N-terminal segment points toward the solvent and
apparently is not involved in protein folding or stability.
Therefore it was predicted that N-terminus mutations would not
alter protein structure or its stability. In agreement with this
prediction all the N-terminus mutants including a mutant protein
carrying deletion of the entire N-terminus .DELTA.2-6 assembled
into a stable complex (FIG. 3). Further, it was surprisingly
uncovered that when the tumor recognition peptides CRGD (SEQ ID
NO:5) and RGDC (SEQ ID NO:6) and RGD loop (SEQ ID NO:10) were
inserted into the SP1 N-terminus, the fusion proteins formed a
boiling stable and protease resistant dodecamer (FIG. 4).
[0276] To confirm the localization of the N-terminus a cysteine
residue was inserted in position 2 of SP1 (Cys 2 variant, SEQ ID
NO:3) (wild type SP1 does not contain any Cys residue). SDS PAGE
analysis of the Cys2 variant shows that Cys2 variant readily forms
intra molecular disulfide bonds within the complex. To confirm the
specificity of the Cys2 insertion we substituted two cysteine
residues located in both Loop 2 (40-44) (SEQ ID NO: 18) and Loop 4
(72-73) (SEQ ID NO: 20) which are also exposed towards the central
cavity. Table 1 shows that in contrast with the Cys 2 SP1 variant,
these mutant proteins fail to form disulfide bonds (under similar
conditions). It should be noted that several recombinant SP1
variants fail to form a soluble protein during expression and form
inclusion bodies (IB). However, it was uncovered that these
inclusion bodies can unfolded with 0.5 M Urea, and refolded by
dialysis (FIG. 5).
[0277] X-ray crystallography studies (see Dgany et al 2004)
indicated numerous putative monomer-monomer and dimer-dimer
interactions stabilize the complex, and it is unlikely that one
amino acid substitution would dramatically destabilize the protein.
Site directed mutagenesis was performed to find the most critical
residues for destabilization of dimer-dimer and monomer-monomer
interactions. Table 1 hereinabove shows that most substitutions did
not destabilized the protein. However, residues I30A (SEQ ID NO:
14), N31A/T34A (SEQ ID NO: 30), F106A (SEQ ID NO:23) and Y108A (SEQ
ID NO:24) (highlighted in Table 1), which are very close to each
other in the protein three dimensional structure, were identified
as hot spots involved in protein stabilization.
[0278] Loop1 (residues 18-22) (SEQ ID NO:6) and L81C (SEQ ID NO:22)
are exposed towards the external perimeters of the ring are good
candidate for multiple presentation of specific peptide involved in
protein-protein interaction as well as interaction with other
molecules or surfaces.
[0279] N-terminus modifications did not effect the protein
structure or stability (for example, dimer formation), but these
modifications provided an opportunity to effect changes in the
binding and complexing characteristics of the SP1 variants (see
Table 1, "N-terminal mutations"), such as complex formation with
metal and metal-associated particles and redox-dependent small
molecule complex formation.
[0280] Some loop 2 modifications (see .DELTA.2-6I40C) can be used
for binding of gold nanoparticles through thiol groups in the
central cavity (Table 1), without significantly altering the
resistant character of the molecule.
[0281] Thus, while not wishing to be limited to a single
hypothesis, while reducing the present invention to practice, the
inventors believe to have uncovered locations and types of
modifications of SP1 molecules resulting in SP1 molecules having
specific, altered properties.
[0282] SP1 Variants can Assemble into Heterodimer:
[0283] In order to determine whether different variant SP1 monomers
can self-assemble to form functional oligomeric complexes,
heterodimer formation between variants was tested.
[0284] In order to produce the heterodimers, the monomeric form of
SP1 was isolated by two methods: electro-elution from SDS PAGE, and
by dissolving inclusion bodies.
[0285] When SP1 variant Cys2loop1RGd is expressed in recombinant
bacteria, the recombinant protein is found in insoluble
proteinaceous inclusion bodies. Solubilization of the SP1 variant
Cys2loop1RGd inclusion bodies by 5M urea results in release of
soluble monomers (see FIG. 5a, lanes 1-4), which are capable of
spontaneously reassembling to the high oligomeric form (FIG. 5b,
lanes 1-4).
[0286] The ability of SP1 to assemble into a hetero-oligomeric
complex was also demonstrated using a similar method. Two variant
recombinant SP1 polypeptides were expressed, a six histidine
N-terminal tagged SP1 (6His2, SEQ ID NO: 7) and an N-terminal
deletion SP1 (.DELTA.NSP1) SEQ ID NO: 2). Monomers of the two SP1
variants were generated by boiling them in the presence of SDS and
separation on preparative SDS-PAGE (FIG. 3, lanes 2 and 3).
Monomers electro-eluted from the gel were mixed to facilitate the
formation of hetero-oligomers (FIG. 3, lanes 4 and 5). To verify
the presence of the two SP1 variants in the self-assembled
hetero-complex, the co-electroeluted protein mixture was further
subjected to nickel affinity column purification (FIG. 3, lanes 6
and 7). Only the histidine tagged protein and its associated
proteins were isolated. The SDS-PAGE analysis showed that, the
6HSP1 complex contains two variants of SP1 (see FIG. 3, lanes 5 and
7). The assembly of hetero-oligomer was finally confirmed by
eliminating the monomeric forms using Proteinase K digestion, (only
the oligomeric form resist the PK digestion) (FIG. 4, lanes 8 and
9).
[0287] These results clearly show that monomers of two SP1 variants
can indeed retain the ability to self-assemble to a hetero-oligomer
complex form.
Example 4
Binding Properties of SP1 Variants
[0288] In order to determine the binding properties of the SP1
variants, and to test the capacity of SP1 and variants to stabilize
molecules complexed thereto, wild type and variant SP1 was exposed
to a variety of biologically active agents, and the resulting
complexes tested for activity, stability and bioavailability of
complexed agents.
[0289] Cys2-SP1 Variant:
[0290] In order to demonstrate the ability to modify and control
complexing of agents to SP1, a Cys2 SP1 variant bearing a cysteine
residue was inserted in position 2 of SP1 (wild type SP1 does not
contain any Cys residue), in the N-terminus which faces the central
cavity (Cys 2 variant). SDS PAGE analysis of the Cys2 variant
(mixed with sample application buffer and subjected to boiling for
10 min in the presence or absence of 2% .beta.-mercaptoethanol)
shows that Cys2 SP1 variant forms disulfide bonds (FIG. 8b).
Reduced Cys2 SP1 variant (treated with 10 mM DTT) is readily
oxidized upon removal of the reducing agent (FIG. 8b). The
specificity of disulfide binding in Cys-2 SP1 is further evidenced
by its failure to react with free sulfhydryl specific reagents such
as 5-5'-Dithio-bis(2-nitrobenzoic acid) (DTNB or Ellman's reagent)
and fluorescein maleimide (data not shown).
[0291] Thus, availability of reactive sulfides in the monomers of
the Cys2 SP1 variant can be controlled by alterations in redox
conditions. In order to determine the effect of redox-dependent
changes on complex formation with agents and other ligands, the
dynamics of Cys 2 SP1 complexing with such agents was investigated.
FIG. 8 shows one possible model for redox-dependent ligand
complexing and release. Without wishing to be limited to one
hypothesis, it is proposed that reducing agents such as the reduced
form of glutathione (GSH), DTT or .beta.-mercaptoethanol can cleave
the disulfide bonds and makes Cys2-SP1 available for complex with
the ligand. The complexed ligand can be bound by oxidation, and can
be dissociated upon reduction FIG. 18 b, and below). This is of
special consideration, since solid tumors are characterized by
hypoxia, and they accumulate large amounts of cellular reducing
equivalents (Kim, 2003). Furthermore it was suggested that high GSH
concentration is involved in drug resistance. Thus, therapeutic or
other agents complexed with Cys2-SP1 under oxidation, can be
stabilized and transported by the SP1 variant, until reaching the
target tissues, whereupon reducing equivalents will enhance release
of the agent(s), as is illustrated in FIGS. 8b and 8c.
[0292] Controlled complexing and release of agents and ligands by
Cys2 Sp1 variants was tested using the complexing of fluoresceine
amine fluorophore (FA) as a water soluble marker ligand, and
doxorubicin (DOX) and paclitaxel (PTX) as models for water soluble
and insoluble therapeutic agents, respectively. Ultrafiltration,
size exclusion chromatography and reverse phase HPLC analysis (see
Methods hereinabove) were used to demonstrate agent-SP1 variant
complexing and release, with respect to the following
considerations:
[0293] 1. Efficiency of ligand complexing by Cys2 SP1 variant
compared to wild type.
[0294] 2. Can protein reduction increase ligand complex formation
by Cys2 SP1 variant but not wild type SP1 complexing?
[0295] 3. Can Cys2 SP1 variant oxidation, prior to ligand addition,
eliminate ligand complexing?
[0296] 4. Can reduction of the Cys2 SP1 variant-ligand complex
increase ligand dissociation?
[0297] 5. Can complexed ligand be stabilized by association with
the Cys2 SP1?
[0298] Fluoresceine Amine (FA):
[0299] FA absorption peak is at 490 nm and at much lower extend at
278 nm (for a given FA concentration OD.sub.278/OD.sub.490=0.19)
(see FIG. 31), when SP1 absorption peak is at 278 nm (see FIG. 30),
and it is not detected at 490 nm. Cys2 SP1 has absorption peaks at
225 nm and 278 nm (see FIG. 32). FA complexing with the Cys2 Sp1
variant was determined following incubation of FA and Cys2 SP1
variant in the absence or presence of GSH, followed by protein
oxidation, ultra filtration (30 kDa cutoff) and exhaustive wash of
the retained fraction with PBS with or without GSH. Absorption
analysis at both 278 and 490 nm. (FIG. 9) shows that protein
reduction with GSH increases complexing of FA and Cys2 SP1, and
that reduction of the FA-Cys2 SP1 complex increases FA
dissociation.
[0300] Size exclusion chromatography (see Methods hereinabove) was
used to compare redox-dependent FA complexing with Cys SP1 variant
and wild type SP1. FIG. 10 shows that wild type SP1 hardly forms
complexes with the FA marker, in the presence or absence of
reducing agents. In contrast, Cys2 SP1 variant complexes with the
FA marker with greater than 3-fold efficiency in the presence of
reducing agents (FIG. 10).
[0301] The superior control of FA complexing with Cys2 SP1 is shown
in FIG. 11. At low ligand concentrations (10 .mu.M), no discernable
FA complexing was detected for wild type SP1, whereas Cys 2 SP1
variant showed efficient complexing. At greater concentrations
(.gtoreq.100 .mu.M), the Cys2 SP1 variant complexes with the FA
marker with an efficiency greater than 3-fold that of wild type
SP1. These results show that Cys2 SP1 variant can efficiently
complex with ligands and agents, compared to wild type and that
protein reduction increases ligand complex formation by the Cys2
SP1 variant, but not the wild type SP1.
[0302] Complex Formation and Covalent Modification of SP1:
[0303] Chemical modification of SP together with a small molecule
allows creation of two types of new complexes: the small molecule
can create both a covalent bond with the protein or a non-covalent
bond by creating a new site for molecular association.
[0304] This was shown with fluorescein amine complexing with SP1
using the carboxylic acid residue modification reagent,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as a covalent
reactive group (Table 5, hereinbelow). Similarly to native SP1, the
SP1-FA complex, is resistant to elevated temperatures. Non-covalent
bound FA dissociates from the protein only upon boiling in the
presence of SDS. Table 5 shows the effect of heat and protease
treatment on fluorescence intensity of SP1-bound FA, showing that
about 80% of fluorescein amine is associated with the protein
through non-covalent association. It is also shown that complexed
SP1-FA retains the characteristic heat-stability and protease
resistance of the wild-type SP1.
TABLE-US-00005 TABLE 5 Stable SP1-FA complex formation using (EDAC)
covalent binding Fluorescence intensity of SP1-FA (% of control)
SP1-FA Treatment conformation Control(none) Protease Heat Oligomer
Complex 100% 90% 95% Monomer 22% 21% 19%
[0305] Doxorubicin (DOX):
[0306] The anthracycline antibiotic doxorubicin is one of the most
useful antineoplastic agents, active against several solid tumors
as well as hematological malignancies. It is used to treat
neoplastic conditions such as acute lymphoblastic leukemia, acute
myeloblastic leukemia, Wilms' tumor, neuroblastoma, soft tissue and
bone sarcomas, breast carcinoma, ovarian carcinoma, transitional
cell bladder carcinoma, thyroid carcinoma, gastric carcinoma,
Hodgkin's disease, malignant lymphoma and bronchogenic carcinoma.
Furthermore, many colloidal carriers of doxorubicin, such as
liposomes and polymeric nanoparticles, have been studied with the
aim of reducing cardiac toxicity and improving therapeutic
efficacy.
[0307] In order to determine the ability of SP1 to form complexes
with DOX, concentrations of both SP1 and the SP1-DOX complex (in
the retained fraction) and free DOX (in the flow-through fraction)
were determined spectroscopically at both 477 and 278 nm. DOX has a
unique optical properties, its absorption peak is at 477 nm, and to
lower extend at 278 nm (for a given DOX concentration
OD.sub.278/OD.sub.477=0.77) (see FIG. 29), while SP1's absorption
peak is at 278 nm, and it is not detected at 477 nm (FIGS. 30 and
32).
[0308] Wild type SP1 and the Cys2 SP1 variant were tested for their
ability to complex with DOX in the presence and absence of DTT, as
determined by ultrafiltration. FIG. 12 shows that DOX forms
complexes with DTT-reduced Cys2 SP1 variant to a much greater
extent that with DTT-treated wild type SP1, and that exposure to
DTT greatly enhances DOX complexing by the Cys2 SP1 variant.
Reduction with glutathione (reduced form) instead of DTT improves
DOX complexing by the Cys2 SP1 variant (data not shown).
[0309] In order to determine whether other manipulations of the
redox state of the chemical environment of the Cys2 SP1 provided
additional control of DOX complexing, DOX complexing to wild type
and Cys2 variant SP1 in the presence or absence of an oxidizing
agent was assessed. FIG. 14 clearly shows that oxidation of Cys2
SP1 variant prior to DOX addition eliminates DOX complexing. Thus,
without wishing to be limited to a single hypothesis, the oxidation
dependent inhibition of DOX-Cys2 SP1 variant complex formation
could indicate that reduction of the disulfide bonds in Cys2 SP1
variant facilitates DOX complex formation.
[0310] In order to further determine the character of Cys2 SP1-DOX
complexing, Cys2 SP1 complex formation with DOX was determined by
size exclusion chromatography at both 475 and 278 nm. FIG. 13 shows
the Cys2 SP1-DOX peak eluting at 7 min, detected at both 278, 475
nm. Extensive wash with PBS, without added reducing agent (FIG. 13,
no GSH) does not disrupt the Cys2 SP1-DOX complex. Addition of a
reducing agent (GSH), however, results in loss of the DOX peak (475
nm) without loss of any of the Cys2 SP1 protein peak (278 nm).
Without wishing to be limited by a single hypothesis, this may be
understood to mean that DOX displays a high affinity to the SP1
complex, and is dissociated from the complex only upon extensive
wash (at low free ligand concentration). Evidence that DOX binds
tightly to the SP1 was also provided by the observation that DOX
does not dissociate from the SP1-DOX complex after 7 days
incubation at room temperature (under oxidizing conditions, data
not shown). It also should be noted that the SP1-DOX complex stays
intact even after ethanol precipitation (see FIG. 35), indicating
high stability of the complex, and ease of removal of free DOX and
the purification of complexed SP1-DOX.
[0311] Analysis of Cys2 SP1-DOX complex by reverse phase HPLC
analysis can be accomplished by eluting the bound species from the
resin (C-18, Pharmacia-Biotech, Uppsala, Sweden) at different
acetonitrile concentrations, and measuring the absorption at both
477 and 278 nm. In order to test the effect of further manipulation
of the redox state on Cys2 SP1 DOX complexing, wild type SP1 and
the Cys2 SP1 variant were tested for their ability to complex with
DOX under reducing conditions (GSH, reduced protein) or oxidizing
conditions (peroxide, oxidized protein). FIG. 14 shows that the
reduced, and not oxidized Cys2 SP1 variant forms complexes with
DOX, and that no complexes are formed between wild type SP1 and
DOX.
[0312] FIG. 14 further shows that free, uncomplexed DOX can be
found in association with the SP1 protein under certain conditions.
Although the samples were extensively washed by ultra-filtration
before application to the column, a significant portion of free DOX
is observed in the reduced (DTT treated) Cys2 SP1 sample,
indicating that some DOX remains associated with the Cys2 SP1, and
it can become dissociated from the protein when subjected to harsh
conditions (75% acetonitrile+0.1% TFA).
[0313] The association of DOX with Cys2 SP1 variant was further
investigated by SDS PAGE analysis, and fluorescence imaging. FIG.
15 shows that the DOX remains associated with all forms of Cys2 SP1
(116 kDa complex, 12.4 kDa monomer and 24.8 kDa dimer), even under
harsh (reduction and boiling) conditions. Further, comparison of
free DOX detected with or without reducing agents (FIG. 15, lanes 2
and 4) shows that complex reduction stimulates DOX released from
the Cys2 SP1-DOX complex. It will be noted the relative intensity
of the bands in this gel does not necessarily reflect the absolute
amount of DOX, possibly due to a self quenching phenomenon. Further
SDS PAGE analysis uncovered that the Cys2 SP1-DOX complex is
resistant to protease (lanes 1-3, 5), heat (85.degree. C./30 min)
(FIG. 16, lanes 1-4) treatments, and incubation in serum
(37.degree. C./24 h) (FIG. 16, lanes 1 and 2). The superior
resistance of the Cys2 SP1-DOX complex to dissociation in harsh
conditions is significant for storage, purification, in-vivo
longevity and other uses of SP1-drug complex.
[0314] Paclitaxel (TAXOL, PTX):
[0315] A common problem in clinical use is the poor solubility of
many drugs. As shown by Dgany et al (Dgany, 2004), the x-ray
crystallography data for SP1 predicts that Helices H1 and H2 define
an external convex surface with numerous hydrophilic and acidic
side chains facing toward the solvent. The inner side of this
surface and the opposing .beta.-sheet enclose a hydrophobic central
cavity rich in aromatic and hydrophobic residues. In order to
determine whether this effects solubilization of small hydrophobic
molecules, SP1 was complexed with Paclitaxel.
[0316] The diterpenoid derivative paclitaxel has broad
antineoplastic activity (ovarian cancer, breast cancer, non-small
cell lung cancer, AIDS-related Kaposi's sarcoma) and a unique
mechanism of action promoting the polymerization and stabilization
of tubulin to microtubules. One of the major clinical problems of
using paclitaxel is its very low solubility in water, due to its
extremely hydrophobic nature. In order to enhance paclitaxel's
solubility, a mixture of 50:50 Cremophor EL (CrEL, a
polyoxyethylated castor oil) and ethanol is used in the current
clinical formulation with serious side effects for 25-30% of
treated patients.
[0317] To circumvent these problems, a great deal of effort has
been directed to developing new systemic paclitaxel formulations,
Cremophor-free with enhanced circulation time. However, none of the
present formulations have overcome the problems.
[0318] In order to determine the stability of the SP1 complex in
organic solvents, SP1 and Cys2 SP1-variant (SEQ ID NO: 3) were
dissolved in organic solvents, dried, reconstituted with aqueous
solvent, and separated on SDS-PAGE. FIG. 18 shows the persistence
of high molecular weight oligomer complexes in both aqueous (Sodium
phosphate, lanes 1 and 4) and organic solvents (lanes 2, 3, 5 and
6). Further, hexane-treated SP1 or Cys2 SP1 variants are resistance
to protease treatment (data not shown).
[0319] Next, the poorly soluble PTX was mixed with lyophilized SP1,
in an organic solvent, in the presence of a reducing agent.
Following evaporation of the organic solvent, the PTX-SP1 complex
was tested for solubility in an aqueous solvent. FIG. 19 shows a RP
HPLC analysis of the enhanced solubility of the SP1-PTX complex, as
compared to free PTX (in DMSO) and uncomplexed SP1. The HPLC
results emphasize the poor solubility of PTX in water (magenta),
with the SP1-PTX complex (red) appearing as the SP1 and PTX peaks
(at 15.8 and 17.4 min, respectively). Ultrafiltration of the
SP1-PTX complex (through a molecular weight cutoff membrane of 30
kDa) shows that the complexed PTX is retained over the membrane,
indicating strong complexing with the SP1.
[0320] PTX does not have a unique absorption spectrum and bound PTX
cannot be detected. PTX dissociates from the SP1-PTX complex in the
presence of high acetonitrile concentrations and it can be detected
in the same elution as free PTX. To demonstrate that all PTX is
eluted from the SP1-PTX complex on the column, PTX was extracted
from the complex using 80% ethanol (FIG. 20), precipitating the SP1
protein. FIG. 20 shows that all PTX can be recovered in solution,
(in contrast with SP1-DOX complex, which precipitates under similar
conditions). At lower ethanol concentrations, PTX can also be
extracted, allowing separation of the complexed SP1-PTX and the
free PTX by ultrafiltration (FIG. 20 and FIG. 21, lower plot).
[0321] In order to determine the efficacy of Cys2 SP1 variant
complex formation with poorly soluble molecules, and the effect of
reducing conditions on the formation of PTX-Cys 2 SP1 variant
complex, PTX was extracted at increasing concentrations of ethanol
from the Cys 2 SP1 variant-PTX complex, in the presence or absence
of 10 mM GSH. FIG. 21 shows that the reducing agent induces PTX
extraction at a lower ethanol concentration. The effect of
reduction on complex formation was also tested. FIG. 22 shows that
the presence of the reducing agent .beta.-ME (12 mM) significantly
enhances Cys2 SP1-PTX complex formation. Unexpectedly, Cys2
SP1-associated PTX was detected in the absence of the reducing
agent, but the associated PTX was removed by filtration.
[0322] These results clearly show that SP1 and SP1 variants form
stable oligomeric complexes in organic as well as aqueous solvents,
that SP1 and SP1 variants can form complexes with poorly soluble
molecules, and that the solubility of poorly water soluble
molecules, such as PTX, is significantly enhanced by association
with the SP1 and SP1 variants. Such solubilization capability of
SP1 has great importance for clinical and other applications of SP1
and SP1 variants, for example, for drug delivery.
[0323] Vinblastine:
[0324] Vinblastine, a vinca alkaloid, is mainly useful for treating
Hodgkin's disease, lymphocytic lymphoma, histiocytic lymphoma,
advanced testicular cancer, advanced breast cancer, Kaposi's
sarcoma. Vinblastine binding to SP1, was determined by Intrinsic
Tryptophan fluorescence measurements.
[0325] SP1 has only one Tryptophan residue (Trp 48). Trp 48 maximal
excitation and emission wavelength are 286 nm and 321 nm
respectively. Upon protein unfolding in 6M Guanidinum HCl, the
maximal emission wavelength is shifted from 321 to 340 nm. FIG. 23
shows that association of SP1 with Vinblastine caused fluorescence
quenching accompanied by a red shift (upon addition of 80 .mu.M
Vinblastine its maximal emission wavelength is shifted to 356 nm).
Unfolded protein fluorescence is also quenched by Vinblastine (FIG.
23), but unlike the native protein it is not accompanied by red
shift. Thus, folded SP1-Vinblastine association can be detected,
and quantified by changes in its Intrinsic Tryptophan
fluorescence.
Example 5
Enhanced Biological Activity of SP1-Associated Drugs
[0326] In order to evaluate the efficacy of SP1 as a drug delivery
agent, or carrier, biological activity of the SP1 and SP1
variant-complexed drug molecules was determined. Thus,
SP1-complexed drug and marker molecules were tested in in-vitro
models and animal models of neoplastic growth.
[0327] SP1-DOX in Colorectal Cancer Cell Line:
[0328] The human colorectal adenocarcinoma cell line, HT-29, was
used to evaluate the biological activity of the SP1-DOX complex,
compared to that of the free drug and un-complexed SP1 protein. The
inhibition concentration 50% (IC.sub.50), defined as the dose of
compound that inhibited 50% of cell growth, for free doxorubicin
and the SP1-DOX complex (prepared by either ultrafiltration or
ethanol precipitation) were similar (FIG. 24a, 0.6 ug/ml), while
uncomplexed protein was inactive. Thus, the SP1-DOX complex is at
least as biologically active as free DOX.
[0329] When the IC.sub.50 values for free PTX (in DMSO) and the
SP1-PTX complex were compared, the value for both preparations were
similar (0.01 ug/ml, FIG. 25), while the unloaded protein (prepared
in parallel to PTX-SP1) was inactive (FIG. 25). However, SP1-PTX
complex remained biologically active ever after at least 3 weeks
storage in aqueous solution, conditions under which free PTX
becomes inactive. Thus, the complexing of PTX with SP1 clearly
increases the stability of the drugs biological activity.
[0330] In order to determine whether SP1-PTX cytotoxicity is
associated with trans-membranal transport of SP1, cells were
exposed to both free and SP1-complexed PTX along with a 10-fold
accesses of uncomplexed SP1. No competition was observed in either
case (data not shown). While not wishing to be limited by a single
hypothesis, it is noted that the absence of competition can
indicate either very fast uptake of the SP1-drug complex by the
cells, or that the drugs dissociate from the complex outside the
cell exert their cytotoxic effects in an uncomplexed manner. The
latter explanation can be associated with high extracellular GSH
concentrations, affecting the redox state of the immediate cellular
environment.
[0331] Biodistribution of SP1 In Vivo:
[0332] To follow the rate of accumulation in a tumor, and the
clearance of administered SP1-complexed molecules from the
circulation, Fluoresceine and SP1-Fluoresceine complex was injected
to C57Bl male mice bearing the B16-F10 (B16) melanoma tumor. 24
hours after the SP1-Fluoresceine injection, the mice were bled, the
animals sacrificed, tumors removed and homogenized in buffer, and
the tissue extracts heat-treated to remove none-specific proteins.
In order to detect accumulation of the SP1-FA complex in the target
tissue, the samples were subjected to SDS-PAGE analysis and
immuno-blot detection with an anti SP1 antibody. FIG. 26 shows that
about 2-5% of injected SP1 complex is found in the tumor, and about
3-15% of injected SP1 remains in circulation 24 hours post
injection, while the free Fluoresceinamine is rapidly cleared.
[0333] Repetitive injections of SP1 to wild type mice were
conducted to demonstrate that SP1 does not induce any significant
immunological response or toxicity. 35 mg/Kg SP1 or PBS control
were injected iv (tail vein) to C57Bl mail mice on days 0, 9, 16,
23, 37 and 53 (6 and 5 animals in the SP1 and PBS groups,
respectively). 55 days past the first injection the animals were
sacrificed and their livers were subjected to histopathology
analysis. Four out of six SP1 treated animals did not show any
pathological response, all through the experiment, up to the 55
days. Histopathology analysis demonstrated that the liver of all
animals from both groups appeared normal. Two out of 6 animals died
after 17 days from an unknown reason. Five out of five PBS treated
animals also showed no pathological response throughout the
experiment. Histological examination of the liver did not show any
signs of pathology.
[0334] In order to determine the degree of immunogenicity of SP1,
anti SP1 antibody production in both PBS- and SP1-treated animals
was detected using ELISA, using either directly immobilized SP1 or
rabbit anti SP1 antibodies (second antibody was HRP conjugated anti
mouse IgG). Serum obtained from both PBS- and SP1-treated animals
had negligible anti-SP1 antibody reaction, with no difference
between the two groups. It should be noted that the rabbit anti-SP1
reacts significantly better with the monomer than with the SP1
oligomer complex, even though the animals were immunized against
the SP1 complex.
[0335] Thus, these results clearly show a biodistribution of SP1
extremely well suited for carrier and drug delivery applications,
and the surprisingly non-toxic and non-immunogenic properties of
SP1 protein in vivo.
[0336] In-Vivo Anti-Tumorogenic Activity of an SP-1-Drug
Complex:
[0337] The effect of SP1-complexing on the anti-tumorogenic
activity of DOX was determined in vivo using the LS147 (human colon
cancer) model in CD1 nude mice (Meyer 1995). Tumor growth rate of
animals receiving SP1-DOX complex or free DOX were (0.5 and 3 mg
DOX/kg, respectively, iv to the tail vein two times a week) was
compared (FIGS. 27a and 27b). This dose (3 mg/Kg) of free DOX
comparable to the maximal tolerate dose in mice. At 35 days past
tumor grafting, the animals were sacrificed, tumors were removed
and their weight recorded (FIGS. 27a and 27b). Since weight loss is
a common side effect of DOX, the animal's weight was also
determined (FIGS. 28a and 28b).
[0338] Although the free DOX dose was 6-fold higher than that of
the SP1-DOX complex dose, the inhibition of tumor growth by SP1-DOX
complex, even at 6 times less concentration than the free DOX, was
comparably significant. In both cases the average tumor size in the
end of the experiment was much smaller than in the PBS-treated
animals. Moreover, histological examination of the tumors showed
extensive necrosis in the DOX and SP1-DOX complex treated
animals.
[0339] However, the DOX-treated animals suffered from serious side
effects, manifested in over 16% weight lose; surprisingly, the
SP1-DOX complex-treated animals did not exhibit any weight
loss.
[0340] Thus, the results brought hereinabove clearly show that
complexing drugs with SP1 enhances important aspects of the drug's
effectiveness, such as solubility and stability in solution, and
can enable reduction in dosage and undesirable side effects,
without concomitant reduction in effectiveness.
Example 6
RP- and Size Exclusion HPLC Profiles of Free and SP1-Complexed
Molecules
[0341] Reverse phase (RP) and size exclusion HPLC were used to
detect and quantitate molecules complexing with SP1 and P1
variants, such as DOX. PXT and FA.
[0342] Size exclusion chromatography is a common method for
separation of molecules of different sizes under mild conditions
and was employed to test FA and DOX complexing with SP1 under mild
conditions. SP1 is eluted from the column after 7 min and is
detected at 278 nm only (FIG. 30), and free FA is eluted from the
column after 16 min and is detected at 490 nm (FIG. 31). The SP1-FA
and SP1-DOX complexes also eluted at the same time, but were
detected at 490 and 475 nm, respectively (FIGS. 29 and 31). FIG. 32
shows a typical SP1 standard curve on size exclusion chromatography
at 278 nm. SP1 is eluted from the column (TSK G3000 SWXL, Tosohaas)
after 7 min and is detected at 278 nm only. FIG. 31 shows
chromatograms of size exclusion chromatography of FA standard
profile at 490 nm. In contrast with free FA, which is eluted from
the column in a distinctive peak, DOX is not eluted in a
distinctive peak (FIG. 29). Although DOX standard curve shows an
absorption peak at 477 nm, this is in reality not available fro
detection. DOX/SP1 ratio can be determined from their standard
curves in solution.
[0343] Reverse phase HPLC (RP-HPLC) analysis also separates between
free ligand and SP1 (see FIGS. 10, 11, 13). This method was used to
test complexing of Doxorubicin and the water insoluble drug
Paclitaxel (PTX). In this case both complexed compounds bound to
the resin (C-18) and were eluted at different acetonitrile
concentrations. FIG. 32 shows the standard profiles of Cys2 SP1
(determined at both 278 and 225 nm). FIG. 33 shows the standard
profile of DOX (determined at 477 nm). FIG. 34 shows the standard
profile for PTX (determined at 225 nm).
[0344] Similar to the results using size exclusion chromatography
the SP1-DOX complexes also eluted at the same time as unloaded SP1
and are detected also at 477 nm, as well as 278 nm. Quantification
of SP1-bound DOX as well as free DOX can be directly calculated
from the absorbance in their peaks at 477 nm because uncomplexed
SP1 does not absorb light at this wave length. Estimation of the
amount of protein in the SP1-DOX peak, at 278 nm can corrected for
DOX absorption at 278 nm according to the following equation
(OD278-0.77*OD.sub.477). In contrast to FA and DOX, PTX does not
display unique absorption properties and complexed PTX cannot be
detected. Apparently PTX dissociates from the SP1-PTX complex in
the presence of high acetonitrile concentrations and it can be
detected in the same elution as free PTX.
[0345] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0346] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 1
1
1521108PRTPopulus tremulamisc_featureWild type Sp1 polypeptide 1Met
Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr Arg 1 5 10
15 Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp
20 25 30 Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe
Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn
Arg Gly Tyr Thr 50 55 60 His Ala Phe Glu Ser Thr Phe Glu Ser Lys
Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala Ala
Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu Val
Ile Asp Tyr Phe Leu Tyr 100 105 2103PRTArtificial sequenceSp1
mutant variant amino acid 2-6 deleted 2Met Lys Leu Val Lys His Thr
Leu Leu Thr Arg Phe Lys Asp Glu Ile 1 5 10 15 Thr Arg Glu Gln Ile
Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu 20 25 30 Asp Leu Ile
Pro Ser Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly 35 40 45 Met
Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser 50 55
60 Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala
65 70 75 80 Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr Leu Ser Gln
Arg Leu 85 90 95 Val Ile Asp Tyr Phe Leu Tyr 100 3109PRTArtificial
sequenceSp1 mutant variant Cys in position 2 3Met Cys Ala Thr Arg
Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr 1 5 10 15 Arg Phe Lys
Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn 20 25 30 Asp
Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe Asn 35 40
45 Trp Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr
50 55 60 Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu
Gln Glu 65 70 75 80 Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu
Gly Phe Leu Pro 85 90 95 Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr
Phe Leu Tyr 100 105 4110PRTArtificial sequenceSp1 mutant variant 2X
His inserted into position 2 4Met His His Ala Thr Arg Thr Pro Lys
Leu Val Lys His Thr Leu Leu 1 5 10 15 Thr Arg Phe Lys Asp Glu Ile
Thr Arg Glu Gln Ile Asp Asn Tyr Ile 20 25 30 Asn Asp Tyr Thr Asn
Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe 35 40 45 Asn Trp Gly
Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly 50 55 60 Tyr
Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln 65 70
75 80 Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe
Leu 85 90 95 Pro Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu
Tyr 100 105 110 5112PRTArtificial sequenceSp1 mutant variant Cys,
Arg, Gly and Asp inserted into position 2 5Met Cys Arg Gly Asp Ala
Thr Arg Thr Pro Lys Leu Val Lys His Thr 1 5 10 15 Leu Leu Thr Arg
Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn 20 25 30 Tyr Ile
Asn Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys 35 40 45
Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn 50
55 60 Arg Gly Tyr Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser
Gly 65 70 75 80 Leu Gln Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe
Ala Glu Gly 85 90 95 Phe Leu Pro Thr Leu Ser Gln Arg Leu Val Ile
Asp Tyr Phe Leu Tyr 100 105 110 6112PRTArtificial sequenceSp1
mutant variant Arg, Gly, Asp and Cys inserted into position 2 6Met
Arg Gly Asp Cys Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr 1 5 10
15 Leu Leu Thr Arg Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn
20 25 30 Tyr Ile Asn Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser
Met Lys 35 40 45 Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser
Ala Glu Leu Asn 50 55 60 Arg Gly Tyr Thr His Ala Phe Glu Ser Thr
Phe Glu Ser Lys Ser Gly 65 70 75 80 Leu Gln Glu Tyr Leu Asp Ser Ala
Ala Leu Ala Ala Phe Ala Glu Gly 85 90 95 Phe Leu Pro Thr Leu Ser
Gln Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100 105 110
7114PRTArtificial sequenceSp1 mutant variant 6X His inserted into
position 2 7Met His His His His His His Ala Thr Arg Thr Pro Lys Leu
Val Lys 1 5 10 15 His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile Thr
Arg Glu Gln Ile 20 25 30 Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu
Leu Asp Leu Ile Pro Ser 35 40 45 Met Lys Ser Phe Asn Trp Gly Thr
Asp Leu Gly Met Glu Ser Ala Glu 50 55 60 Leu Asn Arg Gly Tyr Thr
His Ala Phe Glu Ser Thr Phe Glu Ser Lys 65 70 75 80 Ser Gly Leu Gln
Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala 85 90 95 Glu Gly
Phe Leu Pro Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe 100 105 110
Leu Tyr 8104PRTArtificial sequenceSp1 mutant variant His inserted
into position 2 together with a deletion of amino acid 2-6 8Met His
Lys Leu Val Lys His Thr Leu Leu Thr Arg Phe Lys Asp Glu 1 5 10 15
Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu 20
25 30 Leu Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp Gly Thr Asp
Leu 35 40 45 Gly Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr His
Ala Phe Glu 50 55 60 Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu
Tyr Leu Asp Ser Ala 65 70 75 80 Ala Leu Ala Ala Phe Ala Glu Gly Phe
Leu Pro Thr Leu Ser Gln Arg 85 90 95 Leu Val Ile Asp Tyr Phe Leu
Tyr 100 9103PRTArtificial sequenceSp1 mutant variant Cys inserted
into position 2 together with a deletion of amino acid 2-7 9Met Cys
Leu Val Lys His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile 1 5 10 15
Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu 20
25 30 Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp Gly Thr Asp Leu
Gly 35 40 45 Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr His Ala
Phe Glu Ser 50 55 60 Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr
Leu Asp Ser Ala Ala 65 70 75 80 Leu Ala Ala Phe Ala Glu Gly Phe Leu
Pro Thr Leu Ser Gln Arg Leu 85 90 95 Val Ile Asp Tyr Phe Leu Tyr
100 10108PRTArtificial sequenceSp1 mutant variant E20K modificated
loop1 10Met Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr
Arg 1 5 10 15 Phe Lys Asp Lys Ile Thr Arg Glu Gln Ile Asp Asn Tyr
Ile Asn Asp 20 25 30 Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met
Lys Ser Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala
Glu Leu Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe Glu Ser Thr Phe
Glu Ser Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala
Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln
Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100 105 11110PRTArtificial
sequenceSp1 mutant variant Cys inserted into position 2, K18R
mutated and a Gly inserted into position 19 11Met Cys Ala Thr Arg
Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr 1 5 10 15 Arg Phe Arg
Gly Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile 20 25 30 Asn
Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe 35 40
45 Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly
50 55 60 Tyr Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly
Leu Gln 65 70 75 80 Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala
Glu Gly Phe Leu 85 90 95 Pro Thr Leu Ser Gln Arg Leu Val Ile Asp
Tyr Phe Leu Tyr 100 105 110 12108PRTArtificial sequenceSp1 mutant
variant R23A mutated 12Met Ala Thr Arg Thr Pro Lys Leu Val Lys His
Thr Leu Leu Thr Arg 1 5 10 15 Phe Lys Asp Glu Ile Thr Ala Glu Gln
Ile Asp Asn Tyr Ile Asn Asp 20 25 30 Tyr Thr Asn Leu Leu Asp Leu
Ile Pro Ser Met Lys Ser Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly
Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe
Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu
Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90
95 Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100 105
13108PRTArtificial sequenceSp1 mutant variant D27A mutated 13Met
Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr Arg 1 5 10
15 Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Ala Asn Tyr Ile Asn Asp
20 25 30 Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe
Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn
Arg Gly Tyr Thr 50 55 60 His Ala Phe Glu Ser Thr Phe Glu Ser Lys
Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala Ala
Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu Val
Ile Asp Tyr Phe Leu Tyr 100 105 14108PRTArtificial sequenceSp1
mutant variant I30A mutated 14Met Ala Thr Arg Thr Pro Lys Leu Val
Lys His Thr Leu Leu Thr Arg 1 5 10 15 Phe Lys Asp Glu Ile Thr Arg
Glu Gln Ile Asp Asn Tyr Ala Asn Asp 20 25 30 Tyr Thr Asn Leu Leu
Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp 35 40 45 Gly Thr Asp
Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr 50 55 60 His
Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr 65 70
75 80 Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro
Thr 85 90 95 Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100
105 15108PRTArtificial sequenceSp1 mutant variant N31A mutated
15Met Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr Arg 1
5 10 15 Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Ala
Asp 20 25 30 Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser
Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu
Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe Glu Ser Thr Phe Glu Ser
Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala
Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu
Val Ile Asp Tyr Phe Leu Tyr 100 105 16108PRTArtificial sequenceSp1
mutant variant T34A mutated 16Met Ala Thr Arg Thr Pro Lys Leu Val
Lys His Thr Leu Leu Thr Arg 1 5 10 15 Phe Lys Asp Glu Ile Thr Arg
Glu Gln Ile Asp Asn Tyr Ile Asn Asp 20 25 30 Tyr Ala Asn Leu Leu
Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp 35 40 45 Gly Thr Asp
Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr 50 55 60 His
Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr 65 70
75 80 Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro
Thr 85 90 95 Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100
105 17108PRTArtificial sequenceSp1 mutant variant D38A mutated
17Met Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr Arg 1
5 10 15 Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn
Asp 20 25 30 Tyr Thr Asn Leu Leu Ala Leu Ile Pro Ser Met Lys Ser
Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu
Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe Glu Ser Thr Phe Glu Ser
Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala
Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu
Val Ile Asp Tyr Phe Leu Tyr 100 105 18103PRTArtificial sequenceSp1
mutant variant amino acids 2-6 deleted and I40C mutated 18Met Lys
Leu Val Lys His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile 1 5 10 15
Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu 20
25 30 Asp Leu Cys Pro Ser Met Lys Ser Phe Asn Trp Gly Thr Asp Leu
Gly 35 40 45 Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr His Ala
Phe Glu Ser 50 55 60 Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr
Leu Asp Ser Ala Ala 65 70 75 80 Leu Ala Ala Phe Ala Glu Gly Phe Leu
Pro Thr Leu Ser Gln Arg Leu 85 90 95 Val Ile Asp Tyr Phe Leu Tyr
100 19108PRTArtificial sequenceSp1 mutant variant E68A mutated
19Met Ala Thr Arg Thr Pro Lys Leu Val Lys His Thr Leu Leu Thr Arg 1
5 10 15 Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn
Asp 20 25 30 Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser
Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu
Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe Ala Ser Thr Phe Glu Ser
Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala
Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu
Val Ile Asp Tyr Phe Leu Tyr 100 105 20103PRTArtificial sequenceSp1
mutant variant amino acids 2-6 deleted and E72C mutated 20Met Lys
Leu Val Lys His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile 1 5 10 15
Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu 20
25 30 Asp
Leu Ile Pro Ser Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly 35 40
45 Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser
50 55 60 Thr Phe Cys Ser Lys Ser Gly Leu Gln Glu Tyr Leu Asp Ser
Ala Ala 65 70 75 80 Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr Leu
Ser Gln Arg Leu 85 90 95 Val Ile Asp Tyr Phe Leu Tyr 100
21103PRTArtificial sequenceSp1 mutant variant amino acids 2-6
deleted and S73C mutated 21Met Lys Leu Val Lys His Thr Leu Leu Thr
Arg Phe Lys Asp Glu Ile 1 5 10 15 Thr Arg Glu Gln Ile Asp Asn Tyr
Ile Asn Asp Tyr Thr Asn Leu Leu 20 25 30 Asp Leu Ile Pro Ser Met
Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly 35 40 45 Met Glu Ser Ala
Glu Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser 50 55 60 Thr Phe
Glu Cys Lys Ser Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala 65 70 75 80
Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr Leu Ser Gln Arg Leu 85
90 95 Val Ile Asp Tyr Phe Leu Tyr 100 22103PRTArtificial
sequenceSp1 mutant variant amino acids 2-6 deleted and L81C mutated
22Met Lys Leu Val Lys His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile 1
5 10 15 Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu
Leu 20 25 30 Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp Gly Thr
Asp Leu Gly 35 40 45 Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr
His Ala Phe Glu Ser 50 55 60 Thr Phe Glu Ser Lys Ser Gly Leu Gln
Glu Tyr Cys Asp Ser Ala Ala 65 70 75 80 Leu Ala Ala Phe Ala Glu Gly
Phe Leu Pro Thr Leu Ser Gln Arg Leu 85 90 95 Val Ile Asp Tyr Phe
Leu Tyr 100 23114PRTArtificial sequenceSp1 mutant variant 6Xhis
inserted into position 2 and F106A mutated 23Met His His His His
His His Ala Thr Arg Thr Pro Lys Leu Val Lys 1 5 10 15 His Thr Leu
Leu Thr Arg Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile 20 25 30 Asp
Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser 35 40
45 Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala Glu
50 55 60 Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser Thr Phe Glu
Ser Lys 65 70 75 80 Ser Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala Leu
Ala Ala Phe Ala 85 90 95 Glu Gly Phe Leu Pro Thr Leu Ser Gln Arg
Leu Val Ile Asp Tyr Ala 100 105 110 Leu Tyr 24108PRTArtificial
sequenceSp1 mutant variant Y108A mutated 24Met Ala Thr Arg Thr Pro
Lys Leu Val Lys His Thr Leu Leu Thr Arg 1 5 10 15 Phe Lys Asp Glu
Ile Thr Arg Glu Gln Ile Asp Asn Tyr Ile Asn Asp 20 25 30 Tyr Thr
Asn Leu Leu Asp Leu Ile Pro Ser Met Lys Ser Phe Asn Trp 35 40 45
Gly Thr Asp Leu Gly Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr 50
55 60 His Ala Phe Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu
Tyr 65 70 75 80 Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe
Leu Pro Thr 85 90 95 Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu
Ala 100 105 25114PRTArtificial sequenceSp1 mutant variant 6Xhis
inserted into position 2, N31A and Y108A mutated 25Met His His His
His His His Ala Thr Arg Thr Pro Lys Leu Val Lys 1 5 10 15 His Thr
Leu Leu Thr Arg Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile 20 25 30
Asp Asn Tyr Ile Ala Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser 35
40 45 Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala
Glu 50 55 60 Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser Thr Phe
Glu Ser Lys 65 70 75 80 Ser Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala
Leu Ala Ala Phe Ala 85 90 95 Glu Gly Phe Leu Pro Thr Leu Ser Gln
Arg Leu Val Ile Asp Tyr Phe 100 105 110 Leu Ala 26114PRTArtificial
sequenceSp1 mutant variant 6Xhis inserted into position 2, T50A and
I52A mutated 26Met His His His His His His Ala Thr Arg Thr Pro Lys
Leu Val Lys 1 5 10 15 His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile
Thr Arg Glu Gln Ile 20 25 30 Asp Asn Tyr Ile Asn Asp Tyr Thr Asn
Leu Leu Asp Leu Ile Pro Ser 35 40 45 Met Lys Ser Phe Asn Trp Gly
Ala Asp Ala Gly Met Glu Ser Ala Glu 50 55 60 Leu Asn Arg Gly Tyr
Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys 65 70 75 80 Ser Gly Leu
Gln Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala 85 90 95 Glu
Gly Phe Leu Pro Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe 100 105
110 Leu Tyr 27114PRTArtificial sequenceSp1 mutant variant 6Xhis
inserted into position 2, F106A and Y108A mutated 27Met His His His
His His His Ala Thr Arg Thr Pro Lys Leu Val Lys 1 5 10 15 His Thr
Leu Leu Thr Arg Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile 20 25 30
Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu Asp Leu Ile Pro Ser 35
40 45 Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala
Glu 50 55 60 Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser Thr Phe
Glu Ser Lys 65 70 75 80 Ser Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala
Leu Ala Ala Phe Ala 85 90 95 Glu Gly Phe Leu Pro Thr Leu Ser Gln
Arg Leu Val Ile Asp Tyr Ala 100 105 110 Leu Ala 28114PRTArtificial
sequenceSp1 mutant variant 6Xhis inserted into position 2, S73A and
S75A mutated 28Met His His His His His His Ala Thr Arg Thr Pro Lys
Leu Val Lys 1 5 10 15 His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile
Thr Arg Glu Gln Ile 20 25 30 Asp Asn Tyr Ile Asn Asp Tyr Thr Asn
Leu Leu Asp Leu Ile Pro Ser 35 40 45 Met Lys Ser Phe Asn Trp Gly
Thr Asp Leu Gly Met Glu Ser Ala Glu 50 55 60 Leu Asn Arg Gly Tyr
Thr His Ala Phe Glu Ser Thr Phe Glu Ala Lys 65 70 75 80 Ala Gly Leu
Gln Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala 85 90 95 Glu
Gly Phe Leu Pro Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe 100 105
110 Leu Tyr 29114PRTArtificial sequenceSp1 mutant variant 6Xhis
inserted into position 2, D38A and S75A mutated 29Met His His His
His His His Ala Thr Arg Thr Pro Lys Leu Val Lys 1 5 10 15 His Thr
Leu Leu Thr Arg Phe Lys Asp Glu Ile Thr Arg Glu Gln Ile 20 25 30
Asp Asn Tyr Ile Asn Asp Tyr Thr Asn Leu Leu Ala Leu Ile Pro Ser 35
40 45 Met Lys Ser Phe Asn Trp Gly Thr Asp Leu Gly Met Glu Ser Ala
Glu 50 55 60 Leu Asn Arg Gly Tyr Thr His Ala Phe Glu Ser Thr Phe
Glu Ser Lys 65 70 75 80 Ala Gly Leu Gln Glu Tyr Leu Asp Ser Ala Ala
Leu Ala Ala Phe Ala 85 90 95 Glu Gly Phe Leu Pro Thr Leu Ser Gln
Arg Leu Val Ile Asp Tyr Phe 100 105 110 Leu Tyr 30114PRTArtificial
sequenceSp1 mutant variant 6Xhis inserted into position 2, N31A and
T34A mutated 30Met His His His His His His Ala Thr Arg Thr Pro Lys
Leu Val Lys 1 5 10 15 His Thr Leu Leu Thr Arg Phe Lys Asp Glu Ile
Thr Arg Glu Gln Ile 20 25 30 Asp Asn Tyr Ile Ala Asp Tyr Ala Asn
Leu Leu Asp Leu Ile Pro Ser 35 40 45 Met Lys Ser Phe Asn Trp Gly
Thr Asp Leu Gly Met Glu Ser Ala Glu 50 55 60 Leu Asn Arg Gly Tyr
Thr His Ala Phe Glu Ser Thr Phe Glu Ser Lys 65 70 75 80 Ser Gly Leu
Gln Glu Tyr Leu Asp Ser Ala Ala Leu Ala Ala Phe Ala 85 90 95 Glu
Gly Phe Leu Pro Thr Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe 100 105
110 Leu Tyr 3111PRTArtificial sequenceTumor surface specific
peptide 31Lys Asn Gly Pro Trp Tyr Ala Tyr Thr Gly Arg 1 5 10
328PRTArtificial sequenceTumor surface specific peptide 32Asn Trp
Ala Val Trp Xaa Lys Arg 1 5 339PRTArtificial sequenceTumor surface
specific peptide 33Tyr Xaa Xaa Glu Asp Leu Arg Arg Arg 1 5
348PRTArtificial sequenceTumor surface specific peptide 34Xaa Xaa
Pro Val Asp His Gly Leu 1 5 359PRTArtificial sequenceTumor surface
specific peptide 35Leu Val Arg Ser Thr Gly Gln Phe Val 1 5
369PRTArtificial sequenceTumor surface specific peptide 36Leu Val
Ser Pro Ser Gly Ser Trp Thr 1 5 379PRTArtificial sequenceTumor
surface specific peptide 37Ala Leu Arg Pro Ser Gly Glu Trp Leu 1 5
389PRTArtificial sequenceTumor surface specific peptide 38Ala Ile
Met Ala Ser Gly Gln Trp Leu 1 5 399PRTArtificial sequenceTumor
surface specific peptide 39Gln Ile Leu Ala Ser Gly Arg Trp Leu 1 5
409PRTArtificial sequenceTumor surface specific peptide 40Arg Arg
Pro Ser His Ala Met Ala Arg 1 5 419PRTArtificial sequenceTumor
surface specific peptide 41Asp Asn Asn Arg Pro Ala Asn Ser Met 1 5
429PRTArtificial sequenceTumor surface specific peptide 42Leu Gln
Asp Arg Leu Arg Phe Ala Thr 1 5 439PRTArtificial sequenceTumor
surface specific peptide 43Pro Leu Ser Gly Asp Lys Ser Ser Thr 1 5
446PRTArtificial sequenceTumor surface specific peptide 44Phe Asp
Asp Ala Arg Leu 1 5 456PRTArtificial sequenceTumor surface specific
peptide 45Phe Ser Asp Ala Arg Leu 1 5 466PRTArtificial
sequenceTumor surface specific peptide 46Phe Ser Asp Met Arg Leu 1
5 476PRTArtificial sequenceTumor surface specific peptide 47Phe Val
Asp Val Arg Leu 1 5 486PRTArtificial sequenceTumor surface specific
peptide 48Phe Thr Asp Ile Arg Leu 1 5 496PRTArtificial
sequenceTumor surface specific peptide 49Phe Asn Asp Tyr Arg Leu 1
5 506PRTArtificial sequenceTumor surface specific peptide 50Phe Ser
Asp Thr Arg Leu 1 5 516PRTArtificial sequenceTumor surface specific
peptide 51Pro Ile His Tyr Ile Phe 1 5 526PRTArtificial
sequenceTumor surface specific peptide 52Tyr Ile His Tyr Ile Phe 1
5 536PRTArtificial sequenceTumor surface specific peptide 53Arg Ile
His Tyr Ile Phe 1 5 547PRTArtificial sequenceTumor surface specific
peptide 54Ile Glu Leu Leu Gln Ala Arg 1 5 5510PRTArtificial
sequenceTumor surface specific peptide 55Cys Val Phe Xaa Xaa Xaa
Tyr Xaa Xaa Cys 1 5 10 5612PRTArtificial sequenceTumor surface
specific peptide 56Cys Xaa Phe Xaa Xaa Xaa Tyr Xaa Tyr Leu Met Cys
1 5 10 5713PRTArtificial sequenceTumor surface specific peptide
57Cys Val Xaa Tyr Cys Xaa Xaa Xaa Xaa Cys Tyr Val Cys 1 5 10
5813PRTArtificial sequenceTumor surface specific peptide 58Cys Val
Xaa Tyr Cys Xaa Xaa Xaa Xaa Cys Trp Xaa Cys 1 5 10 598PRTArtificial
sequenceTumor surface specific peptide 59Asp Pro Arg Ala Thr Pro
Gly Ser 1 5 6012PRTArtificial sequenceTumor surface specific
peptide 60His Leu Gln Leu Gln Pro Trp Tyr Pro Gln Ile Ser 1 5 10
6112PRTArtificial sequenceTumor surface specific peptide 61Val Pro
Trp Met Glu Pro Ala Tyr Gln Arg Phe Leu 1 5 10 6212PRTArtificial
sequenceTumor surface specific peptide 62Thr Ser Pro Leu Asn Ile
His Asn Gly Gln Lys Leu 1 5 10 639PRTArtificial sequenceTumor
vascular peptides 63Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5
6411PRTArtificial sequenceTumor vascular peptides 64Ala Cys Asp Cys
Arg Gly Asp Cys Phe Cys Gly 1 5 10 6513PRTArtificial sequenceTumor
vascular peptides 65Cys Asn Gly Arg Cys Val Ser Gly Cys Ala Gly Arg
Cys 1 5 10 669PRTArtificial sequenceTumor vascular peptides 66Cys
Val Cys Asn Gly Arg Met Glu Cys 1 5 676PRTArtificial sequenceTumor
vascular peptides 67Asn Gly Arg Ala His Ala 1 5 6810PRTArtificial
sequenceTumor vascular peptides 68Thr Ala Ala Ser Gly Val Arg Ser
Met His 1 5 10 6910PRTArtificial sequenceTumor vascular peptides
69Leu Thr Leu Arg Trp Val Gly Leu Met Ser 1 5 10 707PRTArtificial
sequenceTumor vascular peptides 70Cys Gly Ser Leu Val Arg Cys 1 5
717PRTArtificial sequenceTumor vascular peptides 71Cys Gly Leu Ser
Asp Ser Cys 1 5 7215PRTArtificial sequenceTumor vascular peptides
72Asn Arg Ser Leu Lys Arg Ile Ser Asn Lys Arg Ile Arg Arg Lys 1 5
10 15 7315PRTArtificial sequenceTumor vascular peptides 73Leu Arg
Ile Lys Arg Lys Arg Arg Lys Arg Lys Lys Thr Arg Lys 1 5 10 15
746PRTArtificial sequenceTumor vascular peptides 74Asn Arg Ser Thr
His Ile 1 5 757PRTArtificial sequenceTumor vascular peptides 75Ser
Met Ser Ile Ala Arg Leu 1 5 767PRTArtificial sequenceTumor vascular
peptides 76Val Ser Phe Leu Glu Tyr Arg 1 5 779PRTArtificial
sequenceTumor vascular peptides 77Cys Pro Gly Pro Glu Gly Ala Gly
Cys 1 5 787PRTArtificial sequenceTumor vascular peptides 78Ala Thr
Trp Leu Pro Pro Arg 1 5 796PRTArtificial sequenceTumor vascular
peptides 79Arg Arg Lys Arg Arg Arg 1 5 8015PRTArtificial
sequenceTumor vascular peptides 80Ala Ser Ser Ser Tyr Pro Leu Ile
His Trp Arg Pro Trp Ala Arg 1 5 10 15 8110PRTArtificial
sequenceTumor vascular peptides 81Cys Thr Thr His Trp Gly Phe Thr
Leu Cys 1 5 10 8214PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 82Met His Gly Lys Thr Gln Ala Thr Ser Gly Thr Ile Gln Ser 1 5
10 8320PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD) 83Ser
Lys Thr Ser Leu Gly Cys Gln Lys Pro Leu Tyr Met Gly Arg Glu 1 5 10
15 Met Arg Met Leu 20 8421PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 84Gln Ala Thr Ser Glu Lys Leu Val Arg
Gly Met Glu Gly Ala Ser Leu 1 5 10 15 His Pro Ala Lys Thr 20
857PRTArtificial sequenceAn inorganic-binding polypeptide selected
by phage display (PD) and cell surface display (CSD) 85Asp Arg Thr
Ser Thr Trp Arg 1 5 867PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 86Gln Ser Val Thr Ser Thr Lys 1
5 877PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD) 87Ser
Ser Ser His Leu Asn Lys 1 5 887PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 88Ser Val Thr Gln Asn Lys Tyr 1 5
897PRTArtificial sequenceAn inorganic-binding polypeptide selected
by phage display (PD) and cell surface display (CSD) 89Ser Pro His
Pro Gly Pro Tyr 1 5 907PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 90His Ala Pro Thr Pro Met Leu 1 5 9112PRTArtificial
sequenceAn inorganic-binding polypeptide selected by phage display
(PD) and cell surface display (CSD) 91Ala Tyr Ser Ser Gly Ala Pro
Pro Met Pro Pro Phe 1 5 10 9212PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 92Asn Pro Ser Ser Leu Phe Arg Tyr Leu
Pro Ser Asp 1 5 10 9312PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 93Ser Leu Ala Thr Gln Pro Pro Arg Thr Pro Pro Val 1 5 10
9412PRTArtificial sequenceAn inorganic-binding polypeptide selected
by phage display (PD) and cell surface display (CSD) 94Met Ser Pro
His Pro His Pro Arg His His His Thr 1 5 10 9512PRTArtificial
sequenceAn inorganic-binding polypeptide selected by phage display
(PD) and cell surface display (CSD) 95Arg Gly Arg Arg Arg Arg Leu
Ser Cys Arg Leu Leu 1 5 10 9612PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 96Lys Pro Ser His His His His His Thr
Gly Ala Asn 1 5 10 9712PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 97Tyr Ser Asp Gln Pro Thr Gln Ser Ser Gln Arg Pro 1 5 10
9812PRTArtificial sequenceAn inorganic-binding polypeptide selected
by phage display (PD) and cell surface display (CSD) 98Thr Tyr His
Ser Ser Gln Leu Gln Arg Pro Pro Leu 1 5 10 9912PRTArtificial
sequenceAn inorganic-binding polypeptide selected by phage display
(PD) and cell surface display (CSD) 99Ser Pro Leu Ser Ile Ala Ala
Ser Ser Pro Trp Pro 1 5 10 10022PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 100Ser Ser Lys Lys Ser Gly Ser Tyr Ser
Gly Tyr Ser Thr Lys Lys Ser 1 5 10 15 Gly Ser Arg Arg Ile Leu 20
10119PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
101Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys Gly Ser Lys Arg
1 5 10 15 Arg Ile Leu 10219PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 102Ser Ser Lys Lys Ser Gly Ser Tyr Ser
Gly Ser Lys Gly Ser Lys Arg 1 5 10 15 Arg Asn Leu 1038PRTArtificial
sequenceAn inorganic-binding polypeptide selected by phage display
(PD) and cell surface display (CSD) 103Ser Ser Arg Cys Ser Ser Ser
Ser 1 5 10412PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
104Val Arg Thr Arg Asp Asp Ala Arg Thr His Arg Lys 1 5 10
10512PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
105Pro Ala Ser Arg Val Glu Lys Asn Gly Val Arg Arg 1 5 10
10620PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
106Asn Thr Arg Met Thr Ala Arg Gln His Arg Ser Ala Asn His Lys Ser
1 5 10 15 Thr Gln Arg Ala 20 1079PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 107Tyr Asp Ser Arg Ser Met Arg Pro His 1
5 10812PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
108Arg His Thr Asp Gly Leu Arg Arg Ile Ala Ala Arg 1 5 10
10912PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
109Arg Thr Arg Arg Gln Gly Gly Asp Val Ser Arg Asp 1 5 10
11012PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
110Arg Pro Arg Arg Ser Ala Ala Arg Gly Ser Glu Gly 1 5 10
11121PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
111Val Lys Thr Gln Ala Thr Ser Arg Glu Glu Pro Pro Arg Leu Pro Ser
1 5 10 15 Lys His Arg Pro Gly 20 11214PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 112Met Asp His Gly Lys Tyr Arg Gln Lys
Gln Ala Thr Pro Gly 1 5 10 11313PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 113His Thr Gln Asn Met Arg Met Tyr Glu
Pro Trp Phe Gly 1 5 10 11413PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 114Asp Val Phe Ser Ser Phe Asn Leu Lys
His Met Arg Gly 1 5 10 1159PRTArtificial sequenceAn
inorganic-binding polypeptide selected by phage display (PD) and
cell surface display (CSD) 115Val Val Arg Pro Lys Ala Ala Thr Asn 1
5 1169PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
116Arg Ile Arg His Arg Leu Val Gly Gln 1 5 1179PRTArtificial
sequenceAn inorganic-binding polypeptide selected by phage display
(PD) and cell surface display (CSD) 117Arg Arg Thr Val Lys His His
Val Asn 1 5 11811PRTArtificial sequenceAn inorganic-binding
polypeptide selected by phage display (PD) and cell surface display
(CSD) 118Ala Gln Asn Pro Ser Asp Asn Asn Thr His Thr 1 5 10
11912PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
119Arg Leu Glu Leu Ala Ile Pro Leu Gln Gly Ser Gly 1 5 10
12012PRTArtificial sequenceAn inorganic-binding polypeptide
selected by phage display (PD) and cell surface display (CSD)
120Thr Pro Pro Arg Pro Ile Gln Tyr Asn His Thr Ser 1 5 10
1217PRTArtificial sequenceAn inorganic-binding polypeptide selected
by phage display (PD) and cell surface display (CSD) 121Asn Asn Pro
Met His Gln Asn 1 5 1226PRTArtificial sequence6XHis tag 122His His
His His His His 1 5 12398PRTTriticum
aestivummisc_feature(50)..(51)Xaa can be any naturally occurring
amino acid 123Val Val Lys His Leu Val Ile Val Gln Phe Lys Glu Asp
Val Thr Pro 1 5 10 15 Glu Arg Leu Asp Gly Leu Ile Arg Gly Tyr Ala
Gly Leu Val Asp Lys 20 25 30 Val Pro Ser Met Lys Ala Phe His Trp
Gly Thr Asp Val Ser Ile Glu 35 40 45 Asn Xaa Xaa Met His Gln Gly
Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60 Glu Ser Thr Glu Gly
Val Lys Glu Tyr Val Tyr His Pro Ala His Val 65 70 75 80 Glu Phe Ala
Thr Asp Phe Leu Gly Ser Thr Glu Lys Val Leu Ile Ile 85 90 95 Asp
Phe 12498PRTTriticum aestivummisc_feature(50)..(51)Xaa can be any
naturally occurring amino acid 124Val Val Lys His Leu Val Ile Val
Gln Phe Lys Glu Asp Val Thr Pro 1 5 10 15 Glu Arg Leu Asp Gly Leu
Ile Arg Gly Tyr Ala Gly Leu Val Asp Lys 20 25 30 Val Pro Ser Met
Lys Ala Phe His Trp Gly Thr Asp Val Ser Ile Glu 35 40 45 Asn Xaa
Xaa Met His Gln Gly Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60
Glu Ser Thr Glu Gly Val Lys Glu Tyr Val Tyr His Pro Ala His Val 65
70 75 80 Glu Phe Ala Thr Asp Phe Leu Gly Ser Thr Glu Lys Val Leu
Ile Ile 85 90 95 Asp Phe 12598PRTTriticum
aestivummisc_feature(50)..(51)Xaa can be any naturally occurring
amino acid 125Val Val Lys His Leu Val Ile Val Gln Phe Lys Glu Asp
Val Thr Pro 1 5 10 15 Glu Arg Leu Glu Gly Leu Ile Arg Gly Tyr Ala
Gly Leu Val Asp Lys 20 25 30 Val Pro Ser Met Lys Ala Phe His Trp
Gly Thr Asp Val Ser Ile Glu 35 40 45 Asn Xaa Xaa Met His Gln Gly
Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60 Glu Ser Thr Glu Gly
Val Lys Glu Tyr Val Tyr His Pro Ala His Val 65 70 75 80 Glu Phe Ala
Thr Asp Phe Leu Gly Ser Thr Glu Lys Val Leu Ile Ile 85 90 95 Asp
Phe 12684PRTZea maysmisc_feature(50)..(51)Xaa can be any naturally
occurring amino acid 126Val Val Lys His Ile Leu Leu Ala Ser Phe Lys
Glu Glu Val Thr Gln 1 5 10 15 Glu Arg Leu Asp Glu Leu Ile Arg Gly
Tyr Ala Ala Leu Val Gly Val 20 25 30 Val Pro Ser Met Lys Ala Phe
His Trp Gly Thr Asp Val Ser Ile Glu 35 40 45 Asn Xaa Xaa Met His
Gln Gly Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60 Glu Ser Thr
Glu Gly Ile Lys Glu Tyr Ile Glu His Pro Ala His Val 65 70 75 80 Glu
Phe Ala Lys 12798PRTOryza sativamisc_feature(50)..(51)Xaa can be
any naturally occurring amino acid 127Val Val Lys His Ile Leu Leu
Ala Arg Phe Lys Glu Asp Val Ala Pro 1 5 10 15 Glu Arg Leu Asp Gln
Leu Ile Arg Gly Tyr Ala Gly Leu Val Asp Leu 20 25 30 Val Pro Ser
Met Lys Ala Phe His Trp Gly Thr Asp Val Ser Ile Glu 35 40 45 Asn
Xaa Xaa Met His Gln Gly Phe Thr His Val Phe Glu Ser Thr Phe 50 55
60 Glu Ser Thr Glu Gly Val Lys Glu Tyr Ile Glu His Pro Ala His Val
65 70 75 80 Glu Phe Ala Asn Glu Phe Leu Pro Val Leu Glu Lys Thr Leu
Ile Ile 85 90 95 Asp Tyr 12898PRTTriticum
aestivummisc_feature(21)..(21)Xaa can be any naturally occurring
amino acid 128Val Val Lys His Leu Val Leu Ala Arg Phe Lys Glu Glu
Ala Thr Pro 1 5 10 15 Glu Ala Leu Asp Xaa Leu Ile Arg Arg Tyr Ala
Gly Leu Val Asp Ala 20 25 30 Val Pro Ser Met Lys Ala Phe His Trp
Gly Thr Asp Val Thr Val Xaa 35 40 45 Xaa Leu Asp Thr His Glu Gly
Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60 Glu Ser Ala Glu Gly
Val Lys Glu Tyr Ile Ala His Pro Ser His Val 65 70 75 80 Glu Phe Val
Asp Glu Phe Leu Ala Leu Ala Glu Lys Met Leu Ile Val 85 90 95 Asp
Tyr 129109PRTArabidopsis thaliana 129Met Glu Glu Ala Lys Gly Pro
Val Lys His Val Leu Leu Ala Ser Phe 1 5 10 15 Lys Asp Gly Val Ser
Pro Glu Lys Ile Glu Glu Leu Ile Lys Gly Tyr 20 25 30 Ala Asn Leu
Val Asn Leu Ile Glu Pro Met Lys Ala Phe His Trp Gly 35 40 45 Lys
Asp Val Ser Ile Glu Asn Leu His Gln Gly Tyr Thr His Ile Phe 50 55
60 Glu Ser Thr Phe Glu Ser Lys Glu Ala Val Ala Glu Tyr Ile Ala His
65 70 75 80 Pro Ala His Val Glu Phe Ala Thr Ile Phe Leu Gly Ser Leu
Asp Lys 85 90 95 Val Leu Val Ile Asp Tyr Lys Pro Thr Ser Val Ser
Leu 100 105 13047PRTArabidopsis thaliana 130Leu His Gln Gly Tyr Thr
His Ile Leu Glu Ser Thr Phe Glu Ser Lys 1 5 10 15 Glu Ala Val Ala
Glu Tyr Ile Ala His Pro Ala His Val Glu Phe Ala 20 25 30 Thr Ile
Phe Leu Gly Ser Leu Asp Lys Val Leu Val Ile Asp Tyr 35 40 45
13198PRTGlycine maxmisc_feature(50)..(51)Xaa can be any naturally
occurring amino acid 131Val Val Lys His Val Leu Leu Ala Lys Phe Lys
Asp Asp Val Thr Pro 1 5 10 15 Glu Arg Ile Glu Glu Leu Ile Lys Asp
Tyr Ala Asn Leu Val Asn Leu 20 25 30 Ile Pro Pro Met Lys Ser Phe
His Trp Gly Lys Asp Val Ser Ala Glu 35 40 45 Asn Xaa Xaa Leu His
Gln Gly Phe Thr His Val Phe Glu Ser Thr Phe 50 55 60 Glu Ser Pro
Glu Gly Val Ala Glu Tyr Val Ala His Pro Ala His Val 65 70 75 80 Glu
Tyr Ala Asn Leu Phe Leu Ser Cys Leu Glu Lys Val Ile Val Ile 85 90
95 Asp Tyr 13298PRTLycopersicon esculentummisc_feature(50)..(51)Xaa
can be any naturally occurring amino acid 132Val Val Lys His Ile
Leu Leu Ala Lys Phe Lys Asp Gly Ile Pro Pro 1 5 10 15 Glu Gln Ile
Asp Gln Leu Ile Lys Gln Tyr Ala Asn Leu Val Asn Leu 20 25 30 Val
Glu Pro Met Lys Ala Phe Gln Trp Gly Lys Asp Val Ser Ile Glu 35 40
45 Asn Xaa Xaa Leu His Gln Gly Phe Thr His Val Phe Glu Ser Thr Phe
50 55 60 Asp Ser Leu Glu Gly Val Ala Glu Tyr Ile Ala His Pro Val
His Val 65 70 75 80 Glu Tyr Ala Asn Thr Leu Leu Pro Gln Leu Glu Lys
Phe Leu Ile Val 85 90 95 Asp Tyr 13393PRTGlycine
maxmisc_feature(49)..(50)Xaa can be any naturally occurring amino
acid 133His Val Leu Leu Pro Lys Leu Lys Asp Tyr Phe Thr Pro Glu Arg
Ile 1 5 10 15 Glu Leu Met Val Asp Tyr Ala Asn Leu Val Asn Leu Met
Pro Arg Met 20 25 30 Lys Ser Phe His Ser Gly Arg Asp Val Ser Ala
Glu Tyr Leu His Leu 35 40 45 Xaa Xaa Gly Cys Thr His Val Tyr Glu
Ser Thr Phe Asp Ser Pro Gly 50 55 60 Val Ala Glu Tyr Val Ala His
Ala Ala His Val Glu Tyr Ala Asn Gln 65 70 75 80 Asp Leu Ser Cys Leu
Glu Lys Val Ile Ala Ile Asp Tyr 85 90 134108PRTPopulus tremula x
Populus tremuloides 134Met Ala Thr Arg Thr Pro Lys Leu Val Lys His
Thr Leu Ala Thr Arg 1 5 10 15 Phe Lys Asp Glu Ile Thr Arg Glu Gln
Ile Asp Asn Tyr Ile Asn Asp 20 25 30 Tyr Thr Asn Leu Leu Asp Leu
Ile Pro Ser Met Lys Ser Phe Asn Trp 35 40 45 Gly Thr Asp Leu Gly
Met Glu Ser Ala Glu Leu Asn Arg Gly Tyr Thr 50 55 60 His Ala Phe
Glu Ser Thr Phe Glu Ser Lys Ser Gly Leu Gln Glu Tyr 65 70 75 80 Leu
Asp Ser Ala Ala Leu Ala Ala Phe Ala Glu Gly Phe Leu Pro Thr 85 90
95 Leu Ser Gln Arg Leu Val Ile Asp Tyr Phe Leu Tyr 100 105
13596PRTTriticum aestivummisc_feature(18)..(20)Xaa can be any
naturally occurring amino acid 135Lys His Leu Cys Leu Val Arg Phe
Lys Glu
Gly Val Val Val Glu Asp 1 5 10 15 Ile Xaa Xaa Xaa Ile Glu Glu Leu
Thr Lys Leu Ala Ala Glu Leu Asp 20 25 30 Thr Val Lys Phe Phe Gly
Trp Gly Lys Asp Val Leu Asn Gln Glu Ala 35 40 45 Xaa Leu Thr Gln
Gly Phe Thr His Val Phe Ser Met Ser Phe Ala Ser 50 55 60 Ala Glu
Asp Leu Ala Ala Tyr Met Gly His Glu Lys His Ser Ala Phe 65 70 75 80
Ala Ala Thr Phe Met Ala Val Leu Asp Lys Val Val Val Leu Asp Phe 85
90 95 13696PRTTriticum aestivummisc_feature(18)..(20)Xaa can be any
naturally occurring amino acid 136Lys His Leu Cys Leu Val Arg Phe
Lys Glu Gly Val Val Val Glu Asp 1 5 10 15 Ile Xaa Xaa Xaa Ile Glu
Glu Leu Thr Lys Leu Ala Ala Glu Leu Asp 20 25 30 Thr Val Lys Phe
Phe Gly Trp Gly Lys Asp Val Leu Asn Gln Glu Ala 35 40 45 Xaa Leu
Thr Gln Gly Phe Thr His Val Phe Ser Met Ser Phe Ala Ser 50 55 60
Ala Glu Asp Leu Ala Ala Cys Met Gly His Glu Lys His Ser Ala Phe 65
70 75 80 Ala Ala Thr Phe Met Ala Val Leu Asp Lys Val Val Val Leu
Asp Phe 85 90 95 13796PRTTriticum aestivummisc_feature(18)..(20)Xaa
can be any naturally occurring amino acid 137Lys His Leu Cys Met
Ala Lys Phe Lys Glu Gly Val Val Val Glu Asp 1 5 10 15 Ile Xaa Xaa
Xaa Ile Gln Glu Leu Thr Lys Leu Ala Ala Glu Leu Asp 20 25 30 Thr
Val Lys Tyr Phe Gly Trp Gly Lys Asp Val Leu Asn Gln Glu Ala 35 40
45 Xaa Leu Thr Gln Gly Phe Thr His Val Phe Val Met Thr Phe Ala Ser
50 55 60 Ala Glu Asp Leu Ala Ala Cys Met Gly His Glu Lys His Thr
Ala Phe 65 70 75 80 Ala Ala Thr Phe Met Ala Ala Leu Asp Lys Val Val
Val Met Asp Phe 85 90 95 13897PRTOryza
sativamisc_feature(15)..(17)Xaa can be any naturally occurring
amino acid 138Val Lys His Leu Cys Leu Val Lys Phe Lys Glu Glu Val
Leu Xaa Xaa 1 5 10 15 Xaa Val Asp Asp Ile Leu Gln Gly Met Thr Lys
Leu Val Ser Glu Met 20 25 30 Asp Met Val Lys Ser Phe Glu Trp Gly
Lys Asp Val Xaa Leu Asn Gln 35 40 45 Glu Met Leu Thr Gln Gly Phe
Thr His Val Phe Ser Leu Thr Phe Ala 50 55 60 Ser Ser Glu Asp Leu
Thr Thr Tyr Met Ser His Glu Arg His Gln Glu 65 70 75 80 Phe Ala Gly
Thr Phe Met Ala Ala Ile Asp Lys Val Val Val Val Asp 85 90 95 Phe
139104PRTSorghum bicolormisc_feature(40)..(42)Xaa can be any
naturally occurring amino acid 139Arg Arg Pro Thr Met Gly Glu Val
Lys His Leu Cys Leu Val Lys Phe 1 5 10 15 Lys Glu Gly Val Val Val
Glu Asp Val Leu Lys Gly Met Thr Asp Leu 20 25 30 Val Ala Gly Met
Asp Met Val Xaa Xaa Xaa Lys Ser Phe Glu Trp Gly 35 40 45 Gln Asp
Val Xaa Leu Asn Gln Glu Met Leu Thr Gln Gly Phe Thr His 50 55 60
Val Phe Ser Leu Thr Phe Ala Phe Ala Asp Asp Leu Ala Thr Tyr Met 65
70 75 80 Gly His Asp Arg His Ala Ala Phe Ala Ala Thr Phe Met Ala
Ala Leu 85 90 95 Asp Lys Val Val Val Ile Asp Phe 100 14077PRTZea
maysmisc_feature(25)..(25)Xaa can be any naturally occurring amino
acid 140Glu Ser Thr Phe Glu Ser Thr Glu Gly Ile Lys Glu Tyr Ile Glu
His 1 5 10 15 Pro Ala His Val Glu Phe Ala Lys Xaa Leu Asn Gln Glu
Met Leu Thr 20 25 30 Gln Gly Phe Thr His Val Phe Ser Leu Thr Phe
Ala Thr Ala Ala Asp 35 40 45 Leu Ala Ala Tyr Met Ala His Asp Ser
His Thr Ala Phe Ala Ala Thr 50 55 60 Phe Met Ala Ala Ile Asp Lys
Val Leu Val Val Asp Phe 65 70 75 14197PRTLycopersicon
esculentummisc_feature(32)..(34)Xaa can be any naturally occurring
amino acid 141Lys His Leu Val Leu Val Lys Phe Lys Glu Asp Val Val
Val Glu Asp 1 5 10 15 Ile Leu Lys Glu Leu Glu Lys Leu Val Gln Glu
Met Asp Ile Val Xaa 20 25 30 Xaa Xaa Lys Ser Phe Val Trp Gly Lys
Asp Val Xaa Xaa Glu Ser His 35 40 45 Glu Met Leu Arg Gln Gly Phe
Thr His Ala Ile Ile Met Thr Phe Asn 50 55 60 Ser Lys Glu Asp Tyr
Gln Thr Phe Ala Asn His Pro Asn His Val Gly 65 70 75 80 Phe Ser Ala
Thr Phe Ala Thr Val Ile Asp Lys Ala Val Leu Leu Asp 85 90 95 Phe
14294PRTSolanum tuberosummisc_feature(29)..(31)Xaa can be any
naturally occurring amino acid 142Leu Leu Val Lys Phe Lys Gln Asp
Val Val Glu Glu Asp Val Leu Lys 1 5 10 15 Gln Ile Glu Gln Leu Val
Asn Glu Ile Asp Leu Ile Xaa Xaa Xaa Lys 20 25 30 Ser Phe Val Trp
Gly Lys Asp Thr Xaa Xaa Glu Ser Asn Glu Met Val 35 40 45 Thr Gln
Gly Tyr Thr His Ala Met Ile Met Thr Phe Asn Ser Lys Glu 50 55 60
Asp Tyr Glu Ala Cys Val Val Lys Glu Val Xaa Xaa Glu Phe Ser Ala 65
70 75 80 Ile Phe Val Thr Val Val Glu Lys Ile Leu Val Leu Asn Phe 85
90 14396PRTGlycine maxmisc_feature(13)..(15)Xaa can be any
naturally occurring amino acid 143His Tyr Val Ile Val Lys Phe Lys
Asp Gly Val Ala Xaa Xaa Xaa Val 1 5 10 15 Asp Asp Leu Ile Gln Gly
Leu Glu Lys Met Val Phe Gly Ile Asp His 20 25 30 Val Lys Ser Phe
Glu Trp Gly Lys Asp Ile Xaa Xaa Glu Ser His Asp 35 40 45 Met Leu
Arg Gln Gly Phe Thr His Ala Phe Leu Met Thr Phe Asn Gly 50 55 60
Lys Glu Glu Phe Asn Ala Phe Gln Thr His Pro Asn His Leu Glu Phe 65
70 75 80 Ser Gly Val Phe Ser Pro Ala Ile Glu Lys Ile Val Val Leu
Asp Phe 85 90 95 14496PRTGlycine maxmisc_feature(13)..(15)Xaa can
be any naturally occurring amino acid 144His Tyr Val Ile Val Lys
Phe Lys Asp Gly Val Ala Xaa Xaa Xaa Val 1 5 10 15 Asp Glu Leu Ile
Gln Gly Leu Glu Lys Met Val Ser Gly Ile Asp His 20 25 30 Val Lys
Ser Phe Glu Trp Gly Lys Asp Ile Xaa Xaa Glu Ser His Asp 35 40 45
Met Leu Arg Gln Gly Phe Thr His Val Phe Leu Met Ala Phe Asn Gly 50
55 60 Lys Glu Glu Phe Asn Ala Phe Gln Thr His Pro Asn His Leu Glu
Phe 65 70 75 80 Thr Gly Val Phe Ser Pro Ala Ile Glu Lys Ile Val Val
Leu Asp Phe 85 90 95 14597PRTGlycine maxmisc_feature(14)..(16)Xaa
can be any naturally occurring amino acid 145Lys His Phe Val Ile
Val Lys Phe Lys Glu Gly Val Ala Xaa Xaa Xaa 1 5 10 15 Val Asp Glu
Leu Thr Lys Gly Met Glu Lys Leu Val Thr Glu Ile Gly 20 25 30 Ala
Val Lys Ser Phe Glu Trp Gly Gln Asp Ile Xaa Xaa Glu Ser Leu 35 40
45 Asp Val Leu Arg Gln Gly Phe Thr His Ala Phe Leu Met Thr Phe Asn
50 55 60 Lys Lys Glu Asp Phe Val Ala Phe Gln Ser His Pro Asn His
Val Glu 65 70 75 80 Phe Ser Thr Lys Phe Ser Ala Ala Ile Glu Asn Ile
Val Leu Leu Asp 85 90 95 Phe 14643PRTGlycine
maxmisc_feature(18)..(19)Xaa can be any naturally occurring amino
acid 146Leu Val Ser Glu Ile His Ala Val Lys Ser Phe Glu Trp Gly Gln
Asp 1 5 10 15 Ile Xaa Xaa Glu Ser Leu Asp Val Leu Arg Gln Gly Phe
Thr His Ala 20 25 30 Phe Leu Met Thr Phe Asn Lys Lys Arg Arg Leu 35
40 147111PRTArabidopsis thaliana 147Met Ala Thr Ser Gly Phe Lys His
Leu Val Val Val Lys Phe Lys Glu 1 5 10 15 Asp Thr Lys Val Asp Glu
Ile Leu Lys Gly Leu Glu Asn Leu Val Ser 20 25 30 Gln Ile Asp Thr
Val Lys Ser Phe Glu Trp Gly Glu Asp Lys Glu Ser 35 40 45 His Asp
Met Leu Arg Gln Gly Phe Thr His Ala Phe Ser Met Thr Phe 50 55 60
Glu Asn Lys Asp Gly Tyr Val Ala Phe Thr Ser His Pro Leu His Val 65
70 75 80 Glu Phe Ser Ala Ala Phe Thr Ala Val Ile Asp Lys Ile Val
Leu Leu 85 90 95 Asp Phe Pro Val Ala Ala Val Lys Ser Ser Val Val
Ala Thr Pro 100 105 110 148100PRTGlycine
maxmisc_feature(39)..(39)Xaa can be any naturally occurring amino
acid 148Lys Thr Val Glu His Ile Val Leu Phe Lys Val Lys Glu Glu Thr
Glu 1 5 10 15 Pro Ser Lys Val Ser Asp Met Val Asn Gly Leu Gly Ser
Leu Val Ser 20 25 30 Leu Asp Pro Val Leu His Xaa Leu Ser Val Gly
Pro Leu Leu Arg Asn 35 40 45 Arg Ser Ser Ala Leu Thr Xaa Xaa Phe
Thr His Met Leu His Ser Arg 50 55 60 Tyr Lys Ser Lys Glu Asp Leu
Glu Ala Tyr Ser Ala His Pro Ser His 65 70 75 80 Val Ser Val Val Lys
Gly Tyr Val Leu Pro Ile Ile Asp Asp Ile Met 85 90 95 Ser Val Asp
Trp 100 149567DNAPopulus tremula 149atccacagag agaaagggaa
gacatggcaa ccagaactcc aaagcttgtg aagcacacat 60tgttgactcg gttcaaggat
gagatcacac gagaacagat cgacaactac attaatgact 120ataccaatct
gctcgatctc attccaagca tgaagagttt caattggggc acggatctgg
180gcatggagtc tgcggagcta aaccgaggat acactcatgc ctttgaatct
acatttgaga 240gcaagtctgg tttgcaagag tacctcgatt ctgctgctct
tgctgcattt gcagaagggt 300ttttgcctac tttgtcacag cgtcttgtga
tagactactt tctctactaa acgctcagga 360gtaacgactt cggccgggct
atttcatggt aataaagtaa tgtaatgttc aataaatgct 420ggttttgaac
cactgaatgt tcgtgtcttg atttcttgtc tgtgctaagt gaagggagtg
480ctgctattcc tttaaaaata aagcccttgg ggttgagttg tagtttttca
atctttttcc 540ccgatttatt tcggtcttgg tgttgtt 567150351DNAArabidopsis
thaliana 150atggaggaag caaagggacc tgtgaagcac gtattgcttg ctagtttcaa
agatggggtt 60agtcctgaga aaatcgaaga gctcatcaaa ggttacgcca atctcgtcaa
tctcatcgaa 120cctatgaaag ctttccactg gggaaaagat gtgagcattg
agaatctgca tcaaggttac 180acacacatct ttgaatccac atttgagagt
aaagaagctg ttgcagagta cattgctcat 240cctgctcacg ttgaattcgc
caccatcttc cttggcagct tggataaagt tttggttatt 300gactacaagc
ctacctctgt ctctctctaa ttatcttgta gcagcatttt c 3511514PRTArtificial
sequenceA tumor specific peptide 151Cys Arg Gly Asp 1
1524PRTArtificial sequenceA tumor specific peptide 152Arg Gly Asp
Cys 1
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