U.S. patent application number 15/877284 was filed with the patent office on 2018-08-02 for peptide having cell membrane penetrating activity.
The applicant listed for this patent is EWHA UNIVERSITY-INDUSTRY COLLABORATION FOUNDATION. Invention is credited to Kyunglim LEE.
Application Number | 20180214565 15/877284 |
Document ID | / |
Family ID | 38437565 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180214565 |
Kind Code |
A1 |
LEE; Kyunglim |
August 2, 2018 |
PEPTIDE HAVING CELL MEMBRANE PENETRATING ACTIVITY
Abstract
Provided are transmembrane complexes that contain a protein
transduction domain (PTD) from the N-terminus of IgE-dependent
histamine-releasing factor (HRF) and a target substance that is to
be delivered into a cell. Also provided are nucleic acid molecules
encoding the transmembrane complex, and methods of delivering the
target substance into a cell interior by contacting the
transmembrane complex with a cell. Also provided are transfection
kits containing the PTD and the target substance.
Inventors: |
LEE; Kyunglim; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EWHA UNIVERSITY-INDUSTRY COLLABORATION FOUNDATION |
Seoul |
|
KR |
|
|
Family ID: |
38437565 |
Appl. No.: |
15/877284 |
Filed: |
January 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15141731 |
Apr 28, 2016 |
9907857 |
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15877284 |
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13669414 |
Nov 5, 2012 |
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15141731 |
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12280077 |
Nov 3, 2008 |
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PCT/KR2007/000885 |
Feb 20, 2007 |
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13669414 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/435 20130101;
A61K 47/64 20170801; C07K 16/18 20130101; A61K 48/0008 20130101;
C07K 7/08 20130101; A61K 47/65 20170801; C07K 14/47 20130101; A61P
43/00 20180101; C07K 14/46 20130101; C07K 7/06 20130101 |
International
Class: |
A61K 47/64 20060101
A61K047/64; C07K 7/08 20060101 C07K007/08; C07K 14/46 20060101
C07K014/46; C07K 14/47 20060101 C07K014/47; C07K 7/06 20060101
C07K007/06; C07K 14/435 20060101 C07K014/435; A61K 47/65 20060101
A61K047/65; A61K 48/00 20060101 A61K048/00; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2006 |
KR |
10-2006-0016156 |
Claims
1. A method for delivering a target substance into a cell,
comprising administering a transmembrane complex to a subject,
wherein the complex comprises: a) a protein transduction domain
(PTD) peptide that consists of the sequence of amino acids set
forth in any of SEQ ID NOS: 39, 41, 43, 45, 26 and 27; and b) a
target substance that is linked to the PTD peptide, wherein: the
target substance is heterologous to the PTD peptide; and the PTD
peptide is linked to the target substance for delivery of the
target substance into the interior of a cell, whereby the target
substance is delivered into the cell.
2. The method of claim 1, wherein the target substance is selected
from among a nucleic acid, a drug, a chemical compound, a
carbohydrate, a lipid, a glycolipid, an enzyme, a regulating
factor, a growth factor and an antibody.
3. The method of claim 1, wherein the PTD peptide is linked to the
target substance via a linker.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/141,731, filed Apr. 28, 2016, which is a divisional of U.S.
application Ser. No. 13/669,414, filed Nov. 5, 2012, which is a
continuation of U.S. application Ser. No. 12/280,077, filed Nov. 3,
2008, which is the U.S. National Stage application of
PCT/KR2007/000885, filed Feb. 20, 2007, which claims priority to
Korean Patent Application No. 10-2006-0016156, filed Feb. 20, 2006,
to Kyunglim Lee, Moonhee Kim, Miyoung Kim and Youngjoo Kwon. The
subject matter of each of the above-mentioned applications is
incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED
ELECTRONICALLY
[0002] An electronic version of the Sequence Listing is filed
herewith, the contents of which are incorporated by reference in
their entirety. The electronic file was created on Jan. 10, 2018,
is 23 kilobytes in size, and titled 380ESEQ.US1.txt.
TECHNICAL FIELD
[0003] The present invention relates to a peptide having cell
membrane penetrating activity, a transmembrane carrier comprising
the peptide having cell membrane penetrating activity as an
effective component, a transmembrane complex consisting of the
peptide having cell membrane penetrating activity combined with a
target substance, a transfection kit comprising the peptide having
cell membrane penetrating activity and the target substance, use of
the peptide having cell membrane penetrating activity for the
manufacture of a transmembrane complex, use of the transmembrane
complex for the manufacture of a medicament, and a method for
delivering a target substance into the cell interior which
comprises administering to a subject a transmembrane complex
consisting of the peptide having cell membrane penetrating activity
combined with a target substance to induce transduction of the
transmembrane complex into the cell interior.
BACKGROUND ART
[0004] Recently, various methods have been developed for delivering
macromolecules such as therapeutic drug, peptides and proteins into
cells in vitro and in vivo.
[0005] In vitro methods include electroporation, membrane fusion
with liposomes, high velocity bombardment with DNA-coated
microprojectiles, incubation with calcium-phosphate-DNA
precipitate, DEAE-dextran mediated transfection, infection with
modified viral nucleic acids, and direct micro-injection into
single cells. But such methods are of extremely limited usefulness
for delivery of proteins.
[0006] Delivery of macromolecules into cells in vivo has been
accomplished with scrape loading, calcium phosphate precipitates
and liposomes. However, these techniques have, up to date, shown
limited usefulness for in vivo cellular delivery.
[0007] General methods for efficient delivery of biologically
active proteins into intact cells, in vitro and in vivo include
chemical addition of a lipopeptide (P. Hoffmann et al., 1988) or a
basic polymer such as polylysine or polyarginine etc. (W-C. Chen et
al., 1978)
[0008] Folic acid has been used as a transport moiety (C. P. Leamon
and Low, 1991). However, these methods have not proved to be highly
reliable or generally useful.
[0009] Recently to introduce macromolecules such as a protein into
a cell interior, gene therapy becomes in the limelight but this
have also problems in that targeting is incorrect. As a
alternative, research on protein transduction or protein therapy is
actively progressed.
[0010] Protein transduction domain (PTD) was first reported that
purified human immunodeficiency virus type-1 ("HIV") TAT protein is
taken up from the surrounding medium by adding it to human cells
growing in culture medium (Green et al., 1988, Frankel et al.,
1988). After this report, drosophila homeotic transcription factor,
antennapedia (Antp) (Joliot et al., 1991) and herpes simplex
virus-1 DNA-binding protein, VP22 (Elliot et al, 1997) were also
identified.
[0011] In comparison of amino acid sequences of the PTDs such as
TAT, Antp and VP22 etc., basic amino acids such as arginine and
lysine exist for the most part (TABLE 1) and this sequence
potentiates easy approach near to the negatively charged
phospholipid bilayer and penetration into the cell interior.
Protein sequences having penetrating activity were named as protein
transduction domains (PTDs).
TABLE-US-00001 TABLE 1 PTD Amino Acid Sequences SEQ ID NO: HW-1 TAT
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg 82 HSV VP22
Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser- 83
Arg-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-
Ser-Arg-Pro-Arg-Arg-Pro-Val-Glu Antp
Arg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys- 84
Trp-Lys-Lys
[0012] In particular, recombinant expression vector was developed
by using a peptide containing 11 amino acids of TAT 47-57 and TAT
fusion proteins were prepared by linking the TAT peptide to other
peptides or proteins and so introduction of full-length protein
into intracellular compartment became possible without the
limitation of size or function (Nagahara et al., 1988).
[0013] As PTDs can be linked with other peptide or proteins to form
fusion protein and then be transduced into cell interior, there are
many attempts to transduce therapeutic drug, peptide, protein etc.
into cell interior using PTDs.
[0014] Recently, it has been known for PTDs which do not contain
lots of basic amino acid residues. Also, it has been reported that
PTDs penetrate phospholipid bilayer of cell membrane by helix
conformation.
[0015] TCTP (translationally controlled tumor protein) is a protein
known as IgE-dependent histamine-releasing factor (HRF) as reported
by MacDonald et al. (1995). TCTP had been known as tumor-specific
protein until 1980' and the synthesis thereof was assumed to be
related to proliferative stage of tumor. TCTP was reported as a
tumor protein of 21 kDa, p21 in mouse erythroleukemia cell line
(Chitpatima et al., 1988). Also, it was revealed that p23, relating
to cell growth in Ehrlich ascites tumor is the same as TCTP/HRF
(Bohm et al, 1989).
[0016] TCTP is frequently found in tumor cell, particularly growing
vigorously, and exists in cytoplasm. It is a known protein
consisting of 172 amino acids (NCBI accession #P13693 (Homo
sapiens)) and shows high homology between species. 45 amino acids
at its C-terminal form basic domain. Because such domain has about
46% homology with MAP-1B, microtubule-associated protein, it was
also assumed that HRF is a microtubule-associated protein. Gachet,
et al. (1997) observed that HRF is distributed consistently along
with the cytoskeletal network to some extent using confocal
microscope, which suggests that HRF binds to the cytoskeleton.
[0017] TCTP expression is characterized by that mRNA is maintained
in regular level, but in case that exterior stimulus such as serum
exists, it is transformed to polysome to be translated. According
to the characteristic, it was named as `Translationally Controlled
Tumor Protein (TCTP)`(Thomas et al., 1981; Thomas and Thomas.,
1986). It was also reported that TCTP mRNA is suppressed during
translation, but when it receives cell division signal, it is
activated and translated to protein (Thomas and Thomas, 1986).
[0018] TCTP/HRF is considered as a histamine releasing material
interacting with basophil or mast cell and related to allergic
inflammatory response.
[0019] MacDonald, et al. (1995) also found that though HRF is an
intracellular protein, HRF in the outside of cells stimulates
IgE-sensitized basophils to release histamine (Schroeder, et al.,
1996). Schroeder, et al. (1997) observed that HRF can augment the
anti-IgE-induced histamine release from all basophils, regardless
of the IgE absence, and thus suggested that HRF exerts its function
by binding to cell membrane receptors, not by binding with IgE.
[0020] The present inventors have previously reported that TCTP/HRF
is interacted with third cytoplasmic domain(CD3) of subunit of
(Na,K)ATPase thereby suppressing the activity of (Na,K)ATPase (as
shown in KR Patent Application No. 10-2001-0027896) (Jung et al.,
2004).
[0021] At the same time the present inventors reports that TCTP/HRF
can pass through cell membrane. Since the amino acid sequence of
TCTP/HRF has no the part consisting of plenty of basic amino acids,
arginine or lysine, which is a characteristic of representative
PTDs, and no similar amino acid sequences to those of other PTDs,
the present inventors considered TCTP has a domain which is
different to other known PTDs in aspect of the protein
structures.
[0022] In whole structure of TCTP, N- and C-terminus get loose and
exposed and middle part forms a spherical shape.
[0023] In prediction of third structure, there are three helixes,
wherein first helix(H1) is very short, second (H2) and third helix
(H3) are exposed to outside. By H2 and H3 structure of TCTP in
Schizosaccharomyces pombe, basic amino acids are distributed to
outside of helix (Thaw et al., 2001) and so H2 and H3 were
predicted to be related to protein transduction activity. However,
by a test result, this helix part had nothing to do with
translocation.
[0024] Therefore if we identify amino acid sequences with protein
transduction function in TCTP/HRF, it may be possible to find new
types of PTD, as well as to make a new drug delivery system though
a novel vector development using these.
[0025] The present inventors made a constant effort for looking for
PTD in TCTP and, as a result, isolated protein transduction domain
composed of very different amino acids in comparison with
well-known PTDs. On the basis of this result, the present inventors
have established the present invention by confirming that this
domain shows remarkably high cell penetrating activity than
well-known PTDs.
DISCLOSURE OF INVENTION
Technical Problem
[0026] It is an object of the present invention to provide a
peptide having cell membrane penetrating activity, a transmembrane
carrier comprising the peptide having cell membrane penetrating
activity as an effective component, a transmembrane complex
consisting of the peptide having cell membrane penetrating activity
combined with a target substance, a transfection kit comprising the
peptide having cell membrane penetrating activity and the target
substance, use of the peptide having cell membrane penetrating
activity for the manufacture of a transmembrane complex, use of the
transmembrane complex for the manufacture of a medicament, and a
method for delivering a target substance into the cell interior
which comprises administering to a subject a transmembrane complex
consisting of the peptide having cell membrane penetrating activity
combined with a target substance to induce transduction of the
transmembrane complex into the cell interior.
Technical Solution
[0027] This invention provides a peptide having cell membrane
penetrating activity, composed of the following amino acid
sequence:
R1-R2-R3-R4-R5-R6-R7-R8-R9-R10
In the above formula, [0028] R1 may be deleted or one amino acid
selected from M, A, Q, C, F, L or W, [0029] R2 may be deleted or
one amino acid selected from I or A, [0030] R3 may be one amino
acid selected from I or A, [0031] R4 may be one amino acid selected
from Y, A, F, S or R, [0032] R5 may be one amino acid selected from
R, A or K, [0033] R6 may be one amino acid selected from D, A, I or
R, [0034] R7 may be deleted or one amino acid selected from L, K,
A, E or R, [0035] R8 may be deleted or one amino acid selected from
I, K or A, [0036] R9 may be deleted or one amino acid selected from
A, S, E, Y or T, [0037] R10 may be deleted or one amino acid
selected from A, H, K or E, and [0038] if R10 is K or H, the amino
acid(s) selected from K, KK, R, RR or HH may be added thereto.
[0039] In one embodiment of the present invention, the amino acid
sequence may be SEQ ID NO:1.
[0040] In one embodiment of the present invention, the amino acid
sequence may be SEQ ID NOS:2-7.
[0041] Also, in one embodiment of the present invention, the amino
acid sequence may be an amino acid sequence which one amino acid of
SEQ ID NO:2 is substituted with alanine. The above amino acid
sequence may be, for example, an amino acid sequence selected from
SEQ ID NOS:8-16, particularly SEQ ID NO:13.
[0042] In addition, in an embodiment of the present invention, the
amino acid sequence may be an amino acid sequence selected from SEQ
ID NOS:20-54. The above sequence may be, for example, an amino acid
sequence selected from SEQ ID NOS:22, 26, 27 or SEQ ID
NOS:31-54.
[0043] In the present invention, `cell membrane penetrating protein
domain` means protein sequence having penetrating activity into
cell interior (cytoplasm, nucleus) across plasma membrane.
[0044] A peptide having cell membrane penetrating activity of the
present invention is a novel cell membrane penetrating protein
domain that has no similarity in sequences with well-known TAT,
VP22 and Antp PTDs(Protein Transduction Domains).
[0045] The present invention provides a peptide having cell
membrane penetrating activity consisting of the amino acid sequence
of SEQ ID NO:1. The present invention also provides a peptide
having cell membrane penetrating activity consisting of one amino
acid sequence selected from SEQ ID NOS:2-7.
[0046] According to one example of the present invention, the
peptide having cell membrane penetrating activity consisting of the
amino acid sequence of SEQ ID NOS:1, 2, 3 or 4 shows excellent cell
penetrating activity in comparison with conventional TAT, and
intracellular penetrating efficiency shows a rapidly increasing
mode when treatment concentration becomes high and incubation time
becomes long.
[0047] In detail, when cell penetrating activity was measured by
using the residues of TCTP from 1.sup.st to 10.sup.th [TCTP(1-10),
SEQ ID NO:1], cell penetrating activity of TCTP(1-10) show over 3
times activity when treated for 15 minutes in 50 .mu.M and 6 times
activity when treated for 15 minutes in 100 .mu.M, compared to that
of TAT. In case of treatment for 2 hours, cell penetrating activity
at concentration of 50 .mu.M and 100 .mu.M of TCTP(1-10) were
higher than those of TAT about 29 times and 30 times,
respectively.
[0048] Also, compared with the case of treatment for 15 minutes,
cell penetrating activity showed an increased fashion in the
incubation time of 2 hours.
[0049] In addition, a peptide comprising amino acid residues of
TCTP(1-9)(SEQ ID NO:2), TCTP(1-8)(SEQ ID NO:3) or TCTP(2-10)(SEQ ID
NO:4) showed more excellent penetrating activity than well-known
TAT(47-58) peptide. Of these, cell penetrating activity was
excellent in the order of TCTP(1-10)(SEQ ID NO:1), TCTP(1-9)(SEQ ID
NO:2), TCTP(1-8)(SEQ ID NO:3) and TCTP(2-10)(SEQ ID NO:4), and when
1st amino acid of TCTP was existing, cell penetrating activity was
more excellent.
[0050] Length of the peptides, as a common length of cell membrane
penetrating protein domain accepted in this art, may vary within
the scope of, preferably, 9-15 residues, and more preferably, 9-10
residues.
[0051] A peptide having cell membrane penetrating activity of the
present invention may be prepared by artificial synthesis or by
isolating the sequence of TCTP(1-10)(SEQ ID NO:1), TCTP(1-9)(SEQ ID
NO:2), TCTP(1-8)(SEQ ID NO:3) or TCTP(2-10)(SEQ ID NO:4) from
TCTP.
[0052] Synthesis of the peptide of the present invention may be
performed, for example, by using an instrument or by using genetic
engineering.
[0053] In case of synthesis by using an instrument, synthesis can
be performed by using Fmoc solid-phase method on automatic peptide
synthesizer (PeptrEX-R48, Peptron). After purifying the synthesized
peptide from resin, the peptide can be purified and analyzed by
reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan) with
Shiseido capcell pak C18 analytic RP column. After synthesis is
completed, the peptide can be identified by a mass spectrometer (HP
1100 Series LC/MSD, Hewlett-Packard, Roseville, USA).
[0054] In case of isolation by genetic engineering, nucleic acid
sequences corresponding to a desired peptide can be introduced into
recombinant vector for protein expression, then the expression of
peptide coding region can be induced by IPTG in E. coli bacteria
like a BL21(.lamda.DE3) or BL21(.lamda.DE3)pLys, that is deficient
in proteases, and the peptide can be purified.
[0055] The present invention also provides a peptide having cell
membrane penetrating activity, composed of the amino acid sequence
of SEQ ID NOS:8-16.
[0056] According to an example of the present invention, among the
amino acid sequences that one amino acid of SEQ ID NO:2 is
substituted with alanine, alanine-substituent of 6th residue,
aspartic acid (SEQ ID NO:13), showed 2.5 times increased
penetrating activity than WT(wild type) peptide at a low
concentration of 10 .mu.M and alanine-substituents of 5.sup.th and
7-9.sup.th residue (R, L, I, S)(SEQ ID NOS:12, 14-16) showed a
little decreased but still showed activity. Activity of
alanine-substituents of 1.sup.st-4.sup.th residues (M, I, I, Y)(SEQ
ID NOS:8-11) was suddenly decreased but maintained functionally
like a WT peptide. Therefore, a peptide having cell membrane
penetrating activity of the present invention comprises the peptide
consisting of one amino acid sequence selected from SEQ ID
NOS:8-16.
[0057] The present invention also provides a peptides having cell
membrane penetrating activity, consisting of one amino acid
sequence selected from SEQ ID NOS:22, 26, 27, or 31-54.
[0058] In an example of the present invention, the peptides of SEQ
ID NOS:20-30 were prepared by deletion, substitution or addition of
one or more amino acids in SEQ ID No.: 1. As a result, the peptides
consisting of SEQ ID NOS:22, 26 or 27 showed better penetrating
activity than TAT (100 .mu.M). On the basis of these penetration
data, the peptides of SEQ ID NOS:31-45 were synthesized repeatedly
and these all peptides showed better penetrating activity than TAT
in 10 .mu.M. On the basis of above data, the peptides of SEQ ID
NOS:46-54 were prepared as various mutant forms of SEQ ID NO:1,
then measured for cell penetrating activity. As a result, the
peptide of SEQ ID NO:49 had excellent activity compared with TAT
and the peptides of SEQ ID NOS:46-54 showed a similar or better
activity compared with TAT and excellent activity compared with
TCTP(1-10)(SEQ ID NO:1). Therefore, a peptide having cell membrane
penetrating activity of the present invention comprises the
peptides consisting of SEQ ID NO:22, 26, 27, or 31-54.
[0059] Length of the peptides, as a common length of cell membrane
penetrating protein domain accepted in this art, may vary within
the scope of, preferably 5-15 residues, and more preferably 8-10
residues.
[0060] The peptide of the present invention may be prepared by
artificial synthesis or by isolating the sequence of TCTP(1-10)(SEQ
ID NO:1), TCTP(1-9)(SEQ ID NO:2), TCTP(1-8)(SEQ ID NO:3) or
TCTP(2-10)(SEQ ID NO:4) and modifying these sequences.
[0061] Synthesis of the peptides may be prepared by same synthesis
methods as described above.
[0062] The present invention also provides a transmembrane carrier
comprising the peptide having cell membrane penetrating activity as
an effective component. The peptide having cell membrane
penetrating activity provides a use as a transmembrane carrier for
penetrating target substance across plasma membrane.
[0063] In addition, the present invention provides a transmembrane
complex consisting of the peptide having cell membrane penetrating
activity combined with a target substance.
[0064] The term `target substance` of the present invention means a
molecule that may be related to a regulation of physiological
activity, made a pharmacological action or maintained a biological
activity in intracellular compartment.
[0065] Target substance of the present invention, for example, may
comprise nucleic acid including DNA and RNA, chemical compound such
as drug, carbohydrate, lipid or glycolipid etc. as non-protein
range molecule, and enzyme, regulation factor, growth factor,
antibody, cytoskeletal factor etc. as protein range molecule.
[0066] A peptide having cell membrane penetrating activity of the
present invention may be linked to one or more target substances by
physically/chemically covalent bond or non-covalent bond, or by
mediators in incorporated or fused forms.
[0067] In detail, if the target substance is a non-protein range
molecule, a peptide having cell membrane penetrating activity of
the present invention may be linked to the target substance by
covalent bond, then the complex may be exposed to target cell
group. In another example, the target substances may be
non-covalently linked to a peptide having cell membrane penetrating
activity of the present invention. For instance, if the target
substance is a nucleic acid, it may be incorporated with a peptide
having cell membrane penetrating activity of the present invention,
in forms of lipid based vehicle, then exposed to target cell
group.
[0068] In case that the target substance is a protein, fusion
protein incorporated with a peptide having cell membrane
penetrating activity of the present invention can be prepared by
obtaining cDNA of the protein(the target substance) through PCR and
cloning cDNA using vectors. If it is impossible, the protein may be
fused chemically. For example, fusion protein can be prepared by
connecting the target substance to linker, then reacting with the
peptide having cell membrane penetrating activity to form
linkage.
[0069] In particular, when the target substance is a protein, the
complex may be penetrated in fauns of fusion protein. In this case,
cell penetrating complex of the present invention may be prepared
as follows.
[0070] First, recombinant expression vector is prepared to generate
a fusion gene encoding a peptide having cell membrane penetrating
activity-target substances conjugate.
[0071] Nucleic acids encoding above fusion protein include the
nucleic acid sequence encoding a peptide having cell membrane
penetrating activity and the nucleic acid sequence encoding a
protein as target substance. For example, these nucleic acid
sequences may comprise sequences consisting of SEQ ID NOS:17-18 or
55-81. Nucleic acid sequences of SEQ ID NOS:17-18 or 55-81 are as
follows.
TABLE-US-00002 Nucleic Acid Sequences SEQ ID Classification (Homo
sapiens) NO: Nucleic acid for SEQ ID NO: 1 (TCTP 1-10)
atgattatctaccgggacctcatcagccac 17 Nucleic acid for SEQ ID NO: 2
(TCTP1-9) atgattatctaccgggacctcatcagc 18 Nucleic acid for SEQ ID
NO: 22 (TCTP-CPP#3) atgattatttttcgcgatctgattagccat 55 Nucleic acid
for SEQ ID NO: 26 (TCTP-CPP#7) atgattatttatcgcgcgctgattagccataaaaaa
56 Nucleic acid for SEQ ID NO: 27 (TCTP-CPP#8)
atgattatttatcgcattgcggcgagccataaaaaa 57 Nucleic acid for SEQ ID NO:
31 (TCTP-CPP#12) atgattatattcgcattgcggcgagccataaaaaa 58 Nucleic
acid for SEQ ID NO: 32 (TCTP-CPP#13)
atgattatttttcgcgcgctgattagccataaaaaa 59 Nucleic acid for SEQ ID NO:
33 (TCTP-CPP#14) atgattatttttcgcgcggcggcgagccataaaaaa 60 Nucleic
acid for SEQ ID NO: 34 (TCTP-CPP#15)
tttattatttttcgcattgcggcgagccataaaaaa 61 Nucleic acid for SEQ ID NO:
35 (TCTP-CPP#16) ctgattatttttcgcattgcggcgagccataaaaaa 62 Nucleic
acid for SEQ ID NO: 36 (TCTP-CPP#17)
tggattatttttcgcattgcggcgagccataaaaaa 63 Nucleic acid for SEQ ID NO:
37 (TCTP-CPP#18) tggattatttttcgcgcggcggcgagccataaaaaa 64 Nucleic
acid for SEQ ID NO: 38 (TCTP-CPP#19)
tggattatttttcgcgcgctgattagccataaaaaa 65 Nucleic acid for SEQ ID NO:
39 (TCTP-CPP#20) atgattatttacgcattgcggcgtatcataaaaaa 66 Nucleic
acid for SEQ ID NO: 40 (TCTP-CPP#21)
tggattatttttcgcattgcggcgtatcataaaaaa 67 Nucleic acid for SEQ ID NO:
41 (TCTP-CPP#22) atgattatttttcgcattgcggcgacccataaaaaa 68 Nucleic
acid for SEQ ID NO: 42 (TCTP-CPP#23)
tggattatttttcgcattgcggcgacccataaaaaa 69 Nucleic acid for SEQ ID NO:
43 (TCTP-CPP#24) atgattatattaaaattgcggcgagccataaaaaa 70 Nucleic
acid for SEQ ID NO: 44 (TCTP-CPP#25)
tggattatttttaaaattgcggcgagccataaaaaa 71 Nucleic acid for SEQ ID NO:
45 (TCTP-CPP#26) atgattatttttgcgattgcggcgagccataaaaaa 72 Nucleic
acid for SEQ ID NO: 46 (TCTP-CPP#27)
ctgattatttttcgcattctgattagccataaaaaa 73 Nucleic acid for SEQ ID NO:
47 (TCTP-CPP#28) atgattatttttcgcattctgattagccataaaaaa 74 Nucleic
acid for SEQ ID NO: 48 (TCTP-CPP#29)
ctgattatttttcgcattctgattagccatcgccgc 75 Nucleic acid for SEQ ID NO:
49 (TCTP-CPP#30) ctgattatttttcgcattctgattagccatcatcat 76 Nucleic
acid for SEQ ID NO: 50 (TCTP-CPP#31)
ctgattatattcgcattctgattagccataaa 77 Nucleic acid for SEQ ID NO: 51
(TCTP-CPP#32) ctgattatttttcgcattctgattagccatcgc 78 Nucleic acid for
SEQ ID NO: 52 (TCTP-CPP#33) ctgattatttttcgcattctgattagccat 79
Nucleic acid for SEQ ID NO: 53 (TCTP-CPP#34)
ctgattatttttgcgattgcggcgagccataaaaaa 80 Nucleic acid for SEQ ID NO:
54 (TCTP-CPP#35) ctgattatttttgcgattctgattagccataaaaaa 81
[0072] Since codons encoding one amino acid are several, nucleic
acid sequences encoding the peptide of the present invention
include all nucleic acid sequence encoding the peptide of the
present invention besides nucleic acid sequences listed in above
table.
[0073] Recombinant expression vector of the present invention may
include conventional promoter for expression, termination factor,
selection marker, reporter gene, tag sequence, restriction enzyme
recognitions site, multi-cloning site and so on.
[0074] Transfection methods to host using recombinant expression
vector of the present invention may be a heat shock or
electroporation etc. which is known in the art.
[0075] After fusion proteins are expressed under proper conditions
in transfected host cell as above, fusion proteins, which consist
of a peptide having cell membrane penetrating activity and a
protein as target substance, may be purified by conventional
methods known in the art.
[0076] In addition, the present invention provides a transfection
kit comprising the peptide having cell membrane penetrating
activity and the target substance. Transfection kits are optimized
systems to introduce easily DNA/RNA to intracellular compartment of
mammalian cell. There are up to now calcium-phosphate method,
methods using lipid complex or dextran complex, but limitations are
that efficiency of these methods is 1/10.sup.6-1/10.sup.2 and
depend on cell type. To overcome these limitations, transfection
kits using the peptide having cell membrane penetrating activity,
may be utilized.
[0077] The transfection kit of the present invention may further
comprise a binding factor combining the peptide with the target
substance. The binding factor means specific DNA/RNA sequences
including transcriptional factor, virus protein, or whole body or a
part of protein that are capable to bind to target substance. For
example, Gal4 is a DNA binding factor. Gal4 is a transcriptional
factor widely used in eukaryote, prokaryote and virus. DNA/RNA
binding factors may be used by vector expressing PTDs and fusion
proteins in vivo and in vitro. Also, incorporation between DNA/RNA
binding factors and PTDs may be accomplished by chemical
interaction, physical interaction or noncovalent interaction.
[0078] If fusion complexes between a peptide having cell membrane
penetrating activity of the present invention and DNA/RNA are
treated outside the cells, it can be overcome both efficiency and
limitation depending on the cell type. Using both a peptide having
cell membrane penetrating activity of the present invention and
DNA/RNA binding factors, it is capable that DNA/RNA is introduced
into cytoplasm and nucleus of various cells in vivo and in vitro.
Particularly, introduction method can be accomplished by various
route including intramuscular, intraperitoneal, intravenous, oral,
subcutaneous, intracutaneous, intranasal introduction and
inhalation.
[0079] In addition, target substance may include one or more
biological regulation substances selected from a group consisting
of protein, lipid, carbohydrate or chemical and transfection kits
of the present invention can introduce above target substance into
cytoplasm and nucleus of various cells in vivo and in vitro. Fusion
between PTD and target substance can be accomplished by chemical,
physical covalent interaction or noncovalent interaction.
[0080] Transfection kit of the present invention provides new
technology about gene therapy and DNA/RNA vaccine according to the
methods of the present invention and can express transiently or
permanently and be used in clinical applications such as gene
therapy and DNA/RNA vaccine as well as basic research.
[0081] Also, the present invention provides a use of the peptide
having cell membrane penetrating activity for the manufacture of a
transmembrane complex and a method for preparing transmembrane
complexes by combining target substance with the peptide having
cell membrane penetrating activity.
[0082] In addition, the present invention provides a use of the
transmembrane complex consisting of the peptide having cell
membrane penetrating activity combined with a target substance for
the manufacture of a medicament and a method for manufacturing a
medicament which comprises mixing the transmembrane complex
consisting of the peptide having cell membrane penetrating activity
combined with a target substance, with a pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier is well
known to a skilled artisan, and the skilled artisan can select and
use the pharmaceutically acceptable carrier which is proper for
introduction to a living body.
[0083] Further, the present invention provides a method for
delivering a target substance into cell interior which comprises
administrating to a subject with a transmembrane complex consisting
of the peptide having cell membrane penetrating activity combined
with a target substance to induce transduction of the transmembrane
complex into cell interior.
[0084] If the target substance is non-protein range molecule, it
may be covalently attached to the peptide having cell membrane
penetrating activity of the present invention, and the complex may
be exposed to target cell group. In another example, the target
substance may be non-covalently attached to the peptide having cell
membrane penetrating activity of the present invention, for
example, if the target substance is a nucleic acid, the complex may
be exposed to target cell group in forms of lipid based vehicle
incorporated with the peptide having cell membrane penetrating
activity of the present invention.
[0085] The `subject` may be mammal including human. The
transmembrane complex can be administrated by various route
including intramuscular, intraperitoneal, intravenous, oral,
subcutaneous, intracutaneous, mucosal administration and
inhalation.
[0086] Dose of the transmembrane complex consisting of the peptide
having cell membrane penetrating activity combined with a target
substance, is variable according to a therapeutically effective
amount of the target substance and penetrating activity of the
peptide, and so it is not limited to a specific dose. Only, for
example, if the target substance is a nucleic acid, the dose of
target substance may be 10.about.1000 .mu.g/kg and the dose of the
peptide of the present invention may be 0.1 mg-10 mg/kg.
[0087] In addition, the present invention provides a method for
treating related diseases by administrating to a subject with the
transmembrane complex consisting of the peptide having cell
membrane penetrating activity combined with a target substance
thereby introducing the target substance into a cell.
[0088] The kind of the disease desired to treatment may be varied
depending on the target substance intended to administrate into
cell interior.
[0089] The `subject` may be mammal including human. The
transmembrane complex can be administrated by various route
including intramuscular, intraperitoneal, intravenous, oral,
subcutaneous, intracutaneous, mucosal administration and
inhalation.
[0090] Also, the present invention provides a nucleic acid sequence
encoding the peptide having cell membrane penetrating activity. For
example, the present invention provides a nucleic acid encoding the
peptide having cell membrane penetrating activity, consisting of an
amino acid sequences selected from SEQ ID NOS:1, 2, 22, 26, 27 or
31-54.
[0091] The nucleic acid may be DNA or RNA of single chain or double
chain and be prepared by synthesizing artificially or isolating
from organism-derived TCTP genes. For example, the nucleic acids
encoding the peptides consisting of SEQ ID NOS:1, 2, 22, 26, 27 or
31-54, represent the nucleic acid sequences of SEQ ID NOS:17-18, or
55-81, respectively.
[0092] Since codons encoding one amino acid are several, nucleic
acid sequences encoding the peptide of the present invention
include all nucleic acid sequences encoding the peptide of the
present invention, and are not limited to the nucleic acid
sequences listed in above table. For example, sequence encoding
alanine in amino acid sequence may be gca, gcc, gcg or get.
[0093] The peptide of the present invention having cell membrane
penetrating activity has a prominent effect in delivery as compared
with TAT-derived peptide. Thus, the peptide having cell membrane
penetrating activity of the present invention, the transmembrane
complex consisting of the peptide combined with a target substance,
and the method for delivering a target substance into a cell using
the transmembrane complex has applications on intracellular
delivery in various research fields as well as on therapeutics of
specific diseases where targeting of drugs is required at high
efficiency. Accordingly, the peptide having cell membrane
penetrating activity of the present invention, the transmembrane
complex consisting of the peptide combined with a target substance,
and the method for delivering a target substance into a cell using
the transmembrane complex is very useful as drug delivery
systems.
[0094] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
ADVANTAGEOUS EFFECTS
[0095] The peptide having cell membrane penetrating activity of the
present invention has a prominent penetrating efficiency as
compared with the activities of prior TAT-derived peptides and so
the peptide has applications in intracellular delivery in various
research fields as well as in therapeutics of specific diseases
where targeting of drugs is required in high efficiency.
Accordingly, the peptide having cell membrane penetrating activity
of the present invention, the transmembrane complex consisting of
the peptide combined with a target substance, and the method for
delivering a target substance into a cell using the transmembrane
complex are very useful as drug delivery systems.
DESCRIPTION OF DRAWINGS
[0096] FIG. 1A and FIG. 1C are schematic diagrams showing various
deletion forms of TCTP of the present invention, and FIG. 1B and
FIG. 1D are the western blot analysis results for cellular uptake
of the various deletion forms of TCTP of FIG. 1A and FIG. 1C in
BEAS-2B cell line.
[0097] FIG. 2 shows a dose dependent cellular uptake after 15
minutes of treatment of TCTP-derived peptides.
[0098] FIG. 3 shows cellular uptake after 2 hours of treatment of
TCTP-derived peptides at various concentrations in HeLa cell
line.
[0099] FIG. 4 shows fluorescence microscope images representing
cellular uptake after 2 hours of treatment of the TCTP-derived
peptides at various concentrations in HeLa cell line.
[0100] FIG. 5 shows cellular uptakes after 2 hours of treatment of
substituents of TCTP-derived peptide at various concentrations at
the sensitivity of 75.
[0101] FIG. 6 shows same result of FIG. 5 at the sensitivity of
100.
[0102] FIG. 7 shows mean fluorescence intensity showing a cellular
uptake of mutant peptides of TCTP-derived peptides(#1-11) treated
for 2 hours at various concentrations using FACS.
[0103] FIG. 8 shows mean fluorescence intensity showing a cellular
uptake of mutant peptides of TCTP-derived peptides(#12-26) treated
for 2 hours at various concentrations using FACS.
[0104] FIG. 9 shows mean fluorescence intensity showing a cellular
uptake of mutant peptides of TCTP-derived peptides(#27-35) treated
for 2 hours at various concentrations using FACS.
[0105] FIG. 10A shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#3, #7, #8) treated for 24 hours at various
concentrations.
[0106] FIG. 10B shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#3, #7, #8) treated for 48 hours at various
concentrations.
[0107] FIG. 11A shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#12-26) treated for 24 hours at various
concentrations.
[0108] FIG. 11B shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#12-26) treated for 48 hours at various
concentrations.
[0109] FIG. 12A shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#27-35) treated for 24 hours at various
concentrations.
[0110] FIG. 12B shows cytotoxicity of mutant peptides of
TCTP-derived peptides (#27-35) treated for 48 hours at various
concentrations.
MODE FOR INVENTION
EXAMPLE 1
Mapping of PTD Using Various Deletion Forms of TCTP
[0111] In order to confirm the region of the TCTP acting as PTD,
various deletion constructs were prepared and then used in the
experiment as follows.
1) Isolation and Purification of Deletion Forms of TCTP
[0112] To overexpress each of those deletion forms of TCTP (FIGS. 1
A and 1C), pRSET vector that is capable of tagging 6 histidine was
employed. Subcloning with DNA sequences corresponding to each
deletion forms of TCTP was performed in the multicloning site of
the vector. Then, the recombinant expression vector was introduced
into E. coli BL21(DE3)(Novagen) or BL21(DE3)pLysS (Novagen). The
expression of the deletion forms of TCTP was induced by IPTG
(isopropyl .beta.-D-thiogalactoside) for 3 hours, then the protein
was isolated and purified by using Ni column which binds to
polyhistidine.
2) Cell Culture and Treatment with the Protein
[0113] BEAS-2B cell was treated with the deletion form of TCTP at
the concentration of 15 .mu.g/ml for 1 hour or 24 hours. Then,
supernatants and cell lysates were obtained and western blotted
with anti-TCTP antibodies (FIG. 1B). As shown in FIG. 1B, full
length TCTP existed in cell supernatants after incubation for 1
hour (Lane 1) but this protein disappeared 24 hours later (Lane 6).
Also, in cell supernatant containing Del-C112HRF lacking
C-terminus, the protein disappeared 24 hours later (Lane 9). On the
other hand, remaining deletion forms of TCTP lacking N-terminus,
Del-N11, N35 and N39C110HRF were still existing in cell supernatant
24 hours later (Lane 7, 8, 10).
[0114] Therefore, it could be known that PDT of TCTP exists in
N-terminus Particularly, since Del-N11HRF was still existed in cell
supernatant 24 hours later (Lane 7), it seems that TCTP 1-10 plays
a role as PDT.
[0115] In addition, it was examined whether TCTP proteins of the
present invention could be transferred to the cellular interior for
a short time, 5 minutes or 30 minutes. The experiment was performed
by same method as the above (FIG. 1D).
[0116] As shown in FIG. 1D, Del-C38HRF holding N-terminus of HRF
disappeared after 30 minutes (Lane 4) in the supernatant while
these proteins were found after 5 minutes (Lane 1) and 30 minutes
(Lane 4) in cell lysates.
[0117] Thus, N-terminus containing TCTP proteins of present
invention can be transferred into cell interior for a short time,
only several minutes to several tens minutes.
EXAMPLE 2
Confirmation of Cell Penetrating Efficiency of the Peptide of the
Present Invention
[0118] As shown in Example 1, in order to confirm that the
N-terminus of TCTP can function as a PTD, the peptides consisting
of N-terminus of TCTP were constructed and examined for cell
penetrating efficiency.
1) Synthesis of Various Peptides Corresponding N-terminus Amino
Acid of TCTP
[0119] TCTP-derived peptides and control peptide, TAT 48-57 were
synthesized as follow.
TABLE-US-00003 Sequence SEQ Classification of amino acid ID NO:
Residues of TCTP(1-10) MIIYRDLISH 1 Residues of TCTP(1-9) MIIYRDLIS
2 Residues of TCTP(1-8) MIIYRDLI 3 Residues of TCTP(2-10) IIYRDLISH
4 Residues of TCTP(1-7) MIIYRDL 5 Residues of TCTP(1-6) MIIYRD 6
Residues of TCTP(3-10) IYRDLISH 7 Control TAT(48-57) GRKKRRQRRR
19
[0120] The N-terminus of each peptide was labeled with fluorescence
dye, rhodamine and the C-terminus was protected. Peptide purity
(>95%) was determined by HPLC. Synthesis of the peptides was
requested to PEPTRON, Inc.
[0121] Negative control was a fluorescence dye, rhodamine
(Molecular Probe) used to label in all peptides.
2) Cell Culture and Incubation of Peptides
[0122] HeLa cell line (ATCC) was propagated in DMEM (GIBCO)
supplemented with 10% FBS (GIBCO) and 100 units/mL
penicillin-streptomycin. Cells were grown in a 5% CO.sub.2
incubator at 37.degree. C.
[0123] HeLa cells were cultured in a 48-well plate until they were
70.about.80% grown up before a day of the experiment. The cells
were washed with DMEM at 37.degree. C. twice, and the TCTP-derived
peptides synthesized in Example 2-1 were treated to the culture
medium in a dose dependent manner (0, 1, 5, 10, 50, 100 .mu.M),
then the cells were incubated for 15 minutes or 2 hours in a
CO.sub.2 incubator at 37.degree. C.
[0124] After the incubation, the cells were washed in cool PBS
three times and immediately measured by a microplate fluorescence
reader (BIO-TEK instruments, Inc., Vermont, USA) at emission 530 nm
and excitation 590 nm for a measurement of rhodamine of
intracellular uptake marker. The sensitivity of reader was set at
100 as a basic mode, but was lowered to 75 if the fluorescent
signals were too strong. All experiments were conducted in triplet
repeats for reproducibility (FIG. 2 and FIG. 3).
[0125] As shown in FIG. 2 and FIG. 3, TAT, control peptide was
transduced into cell in a dose and time-dependent manner as
previously known.
[0126] TCTP (1-10), (1-9), (1-8) peptides of the present invention
were translocated not in 1-10 .mu.M but in 50-100 .mu.M at 15
minutes (FIG. 2) or 2 hours (FIG. 3). In 50-100 .mu.M,
intracellular translocation was observed to be very high and could
not detect due to a strong fluorescence particularly after 2 hours
treatment and thus the sensitivity of reader was lowered to 75.
[0127] In FIG. 3, judging from the fact that there was no
difference on translocation efficiency between 2 hour treatment at
concentration 50 .mu.M and that at 100 .mu.M of TCTP(1-10) peptide,
it seemed that TCTP(1-10) peptide was saturated at 50 .mu.M. On the
other side, TAT (48-57) peptide was saturated at 1 .mu.M or
more.
[0128] TCTP (2-10) peptide was not translocated at a concentrarion
of 1 .mu.M to 10 .mu.M, but was more efficiently translocated at
100 .mu.M after 15 minutes treatment of this peptide. After 2
hours, this peptide has similar cell membrane penetrating activity
to control peptide, TAT(48-57), and was more efficiently
translocated at 100 .mu.M than control peptide.
[0129] So, it could be confirmed that TCTP (1-10), (1-9), (1-8) and
(2-10) peptides having cell membrane penetrating activity of the
present invention had superior ability than well-known PTD, TAT in
their translocation efficiency.
[0130] For TCTP-derived peptide, it had been shown a sudden
increase in translocation ability at the high concentration and
these results might be caused by a difference in translocation
mechanisms.
[0131] Consequently, it could be confirmed that TCTP (1-10), (1-9),
(1-8) and (2-10) peptides having cell membrane penetrating activity
of the present invention had superior ability than well-known PTD,
TAT in their translocation efficiency. From among these peptides,
translocation efficiency was superior in the order of TCTP (1-10),
(1-9), (1-8) and (2-10) peptides, and existence of methionine
(1.sup.st amino acid residue) of TCTP N-terminus was important.
EXAMPLE 3
Identification of Intracellular Translocation of TCTP-Derived
Peptide by Fluorescence Microscope
[0132] The intracellular translocation of the peptide was
identified by fluorescence microscope. HeLa cells were treated with
TCTP (1-9)(SEQ ID NO:2) at a concentration of 10 .mu.M and 100
.mu.M by the same method of Example 2-2. A point of difference was
that HeLa cells were seeded in a 12 well-plate covered a glass
since the plastic plate had a property of fluorescence
interference. After washing, cells on cover glass attached slide
glass were observed (FIG. 4).
[0133] As shown in FIG. 4, the peptide of the present invention was
weakly translocated at a low concentration of 10 .mu.M and strongly
at a high concentration of 100 .mu.M. It was found that the
peptides were distributed widely in the cytoplasm and nucleus of
the cell.
EXAMPLE 4
Identification of Intracellular Translocation of Peptide
Substituents
[0134] In order to confirm that substituent forms of the present
peptide can function as a PTD, substituents of the peptide were
constructed and examined for cell penetrating efficiency.
1) Construction of Peptide Substituents
[0135] Serial substituents of TCTP(1-9)(SEQ ID NO:2) with alanine
were synthesized as follows.
TABLE-US-00004 Sequence SEQ Classification of amino acid ID NO:
TCTP(1-9)M1A AIIYRDLIS 8 TCTP(1-9)I2A MAIYRDLIS 9 TCTP(1-9)I3A
MIAYRDLIS 10 TCTP(1-9)Y4A MIIARDLIS 11 TCTP(1-9)R5A MIIYADLIS 12
TCTP(1-9)D6A MIIYRALIS 13 TCTP(1-9)L7A MIIYRDAIS 14 TCTP(1-9)I8A
MIIYRDLAS 15 TCTP(1-9)S9A MIIYRDLIA 16
[0136] N-terminus of each peptide was labeled with fluorescence
dye, rhodamine and C-terminus was protected. Peptide purity
(>95%) was determined by HPLC. Synthesis of peptides of present
invention was requested to PEPTRON, Inc.
2) Cell Culture and Incubation of Peptides
[0137] HeLa cell line was propagated in DMEM supplemented with 10%
FBS and 100 units/mL penicillin-streptomycin. Cells were grown in a
5% CO.sub.2 incubator at 37.degree. C.
[0138] HeLa cells were cultured in a 48-well plate until they were
70.about.80% grown up before a day of the experiment. The cells
were washed with DMEM at 37.degree. C. twice, and the TCTP-derived
peptides synthesized in Example 4-1 were treated to the culture
medium in a dose dependent manner (0, 1, 10, 100 .mu.M), then the
cells were incubated for 15 minutes or 2 hours in a CO.sub.2
incubator at 37.degree. C.
[0139] After the incubation, the cells were washed in cool PBS
three times and immediately measured by a microplate fluorescence
reader at emission 530 nm and excitation 590 nm for a measurement
of rhodamine of intracellular uptake marker. The sensitivity of
reader was set at 100 as a basic, but was lowered to 75 if
fluorescent signals were strong. All experiments were conducted in
triplet repeats for reproducibility (FIG. 5 and FIG. 6).
[0140] As shown in FIG. 5, when fluorescence intensity of TCTP
(1-9) at 100 .mu.M was set to be 100%, the alanine substituents
showing the largest decline in uptake were alanine substituents for
amino acid residue 1,2,3,4 (each M, I, I, Y) of TCTP(1-9)(each SEQ
ID NOS:8, 9, 10, 11), down by 80-90 percent.
[0141] On the other hand, alanine substituents for amino acid
residue 5, 6, 7, 8, 9 (each R, D, L, I, S) of TCTP(1-9)(each SEQ ID
NOS:12, 13, 14, 15, 16) were declined in uptake, down by about 50
percent but we judged that these peptides were still maintained in
translocation activity. Thus, it was known that four amino acids
(M, I, I, Y) of the N-terminus of TCTP were necessary in cell
penetrating activity.
[0142] Meanwhile, when the sensitivity of KC4 plate reader was set
down to 75, we could not analyze the result of cell penetrating
activity at relatively low concentration of 1 or 10 .mu.M, so
sensitivity of reader was fixed at 100 (FIG. 6). At this time,
because fluorescence intensity at 100 .mu.M was very strong, we
could not express in a same graph.
[0143] As shown in FIG. 6, when fluorescence intensity of TCTP
(1-9) at 10 .mu.M was set to be 1, alanine substituent for amino
acid residue 6th, aspartic acid of TCTP(1-9) (SEQ ID NO:13) had 2.5
times higher penetrating activity than natural peptide, TCTP(1-9).
Aspartic acid is a amino acid with negative charge and only residue
having negative charge of TCTP(1-9). Thus it was considered that
amino acid with negative charge decreased the activity of cell
penetration of TCTP.
[0144] Natural peptides of TCTP(1-10), (1-9), (1-8), (2-10) were
efficiently translocated at a high concentration, while these
peptides had lower efficiency than control peptide, TAT at a
relatively low concentration of 1 .mu.M and 10 .mu.M (EXAMPLE 2).
However, from the above results it was shown that analogues of
deletion, addition or substitution of 6th residue had a excellent
penetrating activity at a low concentration.
[0145] From all of the above results, four amino acids(M, I, I, Y)
on N-terminus of TCTP played a necessary role in cell penetrating
activity and particularly alanine substituent for 6th residue,
aspartic acid increased suddenly cell penetrating activity at a low
concentration (10 .mu.M). At this time, we assumed that which
penetrating activity was increased at a low concentration but
decreased at a high concentration was due to low solubility of
alanine substituent with hydrophobic property.
EXAMPLE 5
Cell Penetrating Activity of Mutant Peptides
[0146] As shown in EXAMPLE 4, it was confirmed that substituent
peptides of the present invention had a cell membrane penetrating
activity. So to identify which mutant forms of the present peptides
have penetrating activity, we examined translocation efficiency of
mutant peptides.
1) Construction of Mutant Peptides
[0147] From the results of EXAMPLE 4, various mutant peptides were
constructed with the frame of TCTP (1-10)(SEQ ID NO:1).
TABLE-US-00005 Sequence SEQ Classification of amino acid ID NO:
TCTP-CPP#1 MIIYRDLISKK 20 TCTP-CPP#2 MIIYRDKKSH 21 TCTP-CPP#3
MIIFRDLISH 22 TCTP-CPP#4 MIISRDLISH 23 TCTP-CPP#5 QIISRDLISH 24
TCTP-CPP#6 CIISRDLISH 25 TCTP-CPP#7 MIIYRALISHKK 26 TCTP-CPP#8
MIIYRIAASHKK 27 TCTP-CPP#9 MIIRRDLISE 28 TCTP-CPP#10 MIIYRAEISH 29
TCTP-CPP#11 MIIYARRAEE 30 TCTP-CPP#12 MIIFRIAASHKK 31 TCTP-CPP#13
MIIFRALISHKK 32 TCTP-CPP#14 MIIFRAAASHKK 33 TCTP-CPP#15
FIIFRIAASHKK 34 TCTP-CPP#16 LIIFRIAASHKK 35 TCTP-CPP#17
WIIFRIAASHKK 36 TCTP-CPP#18 WIIFRAAASHKK 37 TCTP-CPP#19
WIIFRALISHKK 38 TCTP-CPP#20 MIIFRIAAYHKK 39 TCTP-CPP#21
WIIFRIAAYHKK 40 TCTP-CPP#22 MIIFRIAATHKK 41 TCTP-CPP#23
WIIFRIAATHKK 42 TCTP-CPP#24 MIIFKIAASHKK 43 TCTP-CPP#25
WIIFKTAASHKK 44 TCTP-CPP#26 MIIFAIAASHKK 45 TCTP-CPP#27
LIIFRILISHKK 46 TCTP-CPP#28 MIIFRILISHKK 47 TCTP-CPP#29
LIIFRILISHRR 48 TCTP-CPP#30 LIIFRILISHHH 49 TCTP-CPP#31 LIIRILISHK
50 TCTP-CPP#32 LIIFRILISHR 51 TCTP-CPP#33 LIIFRILISH 52 TCTP-CPP#34
LIIFAIAASHKK 53 TCTP-CPP#35 LIIFAILISHKK 54
[0148] N-terminus of each peptide was labeled with fluorescence
dye, FITC and C-terminus was protected. Peptide purity (>95%)
was determined by HPLC. Synthesis of the peptides of the present
invention was requested to PEPTRON, Inc.
2) Cell Culture and Incubation of Peptides
[0149] HeLa cell line was propagated in DMEM supplemented with 10%
FBS and 100 units/mL penicillin-streptomycin. Cells were grown in a
5% CO.sub.2 incubator at 37.degree. C.
[0150] HeLa cells were cultured in a 6-well plate until they were
70.about.80% grown up before a day of the experiment. The cells
were washed with DMEM at 37.degree. C. twice, and the TCTP-derived
peptides synthesized in Example 5-1 were treated to the culture
medium in a dose dependent manner (0, 1, 10, 100 .mu.M), then the
cells were incubated for 2 hours in a CO.sub.2 incubator at
37.degree. C.
[0151] After the incubation, the cells were washed in cool PBS two
times and treated with 1 mg/ml trypsin for 15 min at 37.degree. C.
to digest peptides attached on cell membrane and washed in PBS
twice again. Then, the cells were analyzed by FACS at emission 510
nm and excitation 530 nm for a measurement of FITC of intracellular
uptake marker (FIGS. 7, 8 and 9). Intracellular translocation
efficiency of mutant peptides, TCTP-CPP#1-35 (SEQ ID NOS:20-54) was
compared to wild type(WT), TCTP(1-10)(SEQ ID NO:1) and control
peptide, TAT(48-57).
3) Relationship Between Peptide Variants and Cell Penetrating
Activity
[0152] When mutant peptides were designed, each position of the
residues can be substituted with all 20 amino acids like alanine
substitution, but this is inefficient to search the best effective
mutant out of all peptides because charge and isoelectric point of
whole peptide after change of other neighboring position of amino
acid also have to be considered. Thus we tried new modification on
the basis of the results deduced after primary changes then we
designed new variant peptides to verify the role of crucial amino
acid. New mutant peptides and sequences were arranged in the table
at EXAMPLE 5-1. We intended to explain the mutated position easily
by giving a number from I to X (from N-terminus) to each ten amino
acid of wild type(WT) (SEQ ID NO:1). To increase the solubility and
binding efficiency of WT to cell membrane(in the same reason of use
of polyarginine and polylysine), we did the lysine substitution at
the position of WT-X and simultaneous addition of lysine at the
same position (SEQ ID NO:20), two lysine substitutions at the
position of WT-VILVIII (SEQ ID NO:21) and two lysine additions to
WT (SEQ ID NO:26)(SEQ ID NO:27). Only SEQ ID NO:26 and SEQ ID NO:27
of these variants increased cell penetrating activity. According to
results comparing and analyzing mean fluorescence intensity (MFI)
when MFI of WT at the concentration of 10 .mu.M was set to 1, TAT,
SEQ ID NO:26 and SEQ ID NO:27 were 6.1 times, 6.04 times and 1.73
times higher than WT at the concentration of 10 .mu.M,
respectively, and TAT, SEQ ID NO:26 and SEQ ID NO:27 were 94.75
times, 144.6 times and 342.9 times higher than WT at the
concentration of 100 .sub.jiM in cell penetrating activity,
respectively. Therefore variant peptides of all 12 amino acids
adding two lysines at C-terminus of WT was maintained in next
designed variant peptides(from SEQ ID NO:31) and substitution with
other basic amino acids than lysine and change of number of basic
amino acids were tested(SEQ ID NOS:48-52). As a result, additions
of 1 or 2 basic amino acid at the C-terminus showed higher
efficiency than WT.
[0153] To analyze the role of sulfur of methionine in the position
of WT-I, we substituted methionine(M) with glutamine(Q) or
cysteine(C)(comparison with SEQ ID NO:23 and SEQ ID NOS:24-25). As
a result, sulfur didn't play a crucial role and so to test the role
of hydrophobicity of methionine, methionine was substituted by
phenylalanine(F), leucine(L) or tryptophan(W) (comparison with SEQ
ID NO:31 and SEQ ID NOS:34-36, comparison with SEQ ID NO:32 and SEQ
ID NO:38, comparison with SEQ ID NO:33 and SEQ ID NO:37, comparison
with SEQ ID NO:39 and SEQ ID NO:40, comparison with SEQ ID NO:41
and SEQ ID NO:42, comparison with SEQ ID NO:43 and SEQ ID NO:44,
comparison with SEQ ID NO:46 and SEQ ID NO:47). Consequently, cell
penetrating activities of SEQ ID NOS:37, 38 and 39 were lower than
SEQ ID NO:34 at the concentration of 100 .mu.M but were 52.0 times,
55.6 times and 25.0 times higher than WT in the concentration of 10
.mu.M, respectively, and so these peptides had an excellent
translocation efficiency in comparison with SEQ ID NO:31 (29 times
higher than WT). As results of SEQ ID NO:38 in comparison with SEQ
ID NO:32 and SEQ ID NO:37 in comparison with SEQ ID NO:33,
substitution for tryptophan did not increase translocation
efficiency. This result might be related to cytotoxicity of
tryptophan substituents at the concentration of 100 .mu.M (FIGS.
11A and 11B). In comparison between SEQ ID NOS:39 and 40, SEQ ID
NOS:41 and 42, SEQ ID NOS:43 and 44, substitution for tryptophan
instead of methionine did not induce the important changes in the
aspect of efficiency and cytotoxicity. Substitution for
phenylalanine (SEQ ID NO:34) or leucine (SEQ ID NO:35) brought
about the increased result of translocation efficiency at the
concentration of 10 .mu.M and a decreased result at 100 .mu.M,
compared to SEQ ID NO:31. Leucine substituents in SEQ ID NOS:31,
34, 35 and 36 caused the most increased result at 10 .mu.M and the
little decreased result at 100 .mu.M. Cytotoxicity of SEQ ID NO:35
was weaker than SEQ ID NO:31 at 100 .mu.M. In SEQ ID NO:46 (3.75
times higher than MFI of WT 10 .mu.M) and SEQ ID NO:47 (7.04 times
higher than MFI of WT 10 .mu.M), substitution for leucine caused
the decreased penetrating activity but toxicity of SEQ ID NO:46 was
weaker than that of SEQ ID NO:47. Considering problems of
methionine with cytotoxicity and reduction instability, we judged
it was most appropriate that methionine was substituted by leucine
and so introduced leucine in peptide variants after this experiment
(From SEQ ID NO:48).
[0154] To test the role of tyrosine(Y) at the position of WT-IV, by
substituting tyrosine with phenylalanine(F) having no hydroxyl
group but isostericity like a tyrosine or serine(S) having hydroxyl
group like a tyrosine, we tested the importance of hydrophobicity
and the action of hydroxyl group and so on in this position. SEQ ID
NOS:22 and 25 were 19.63 times and 0.91 times higher than WT at 10
.mu.M and 216.75 times and 1.81 times higher at 100 .mu.M,
respectively. From this result, it was known that increase of
hydrophobicity enhanced cell penetrating activity in this position,
and so after this experiment we introduced phenylalanine in the
position of WT-IV of peptide variants (From SEQ ID NO:31).
[0155] We compared substituents for basic amino acid by
substituting arginine(R) with lysine(comparison between SEQ ID
NOS:31 and 43, and between SEQ ID NOS:36 and 44) or alanine
(comparison between SEQ ID NOS:31 and 45 and between SEQ ID NOS:35
and 53) in the position of WT-V. As a result, translocation
efficiency of SEQ ID NO:31 (26.77 times increase in comparison with
WT) was lower than SEQ ID NO:43 (12.1 times increase) and
efficiency of SEQ ID NO:36 (18.4 times increase in comparison with
WT) was lower than SEQ ID NO:44 (15.04 times increase) at 10 .mu.M.
Translocation efficiency of SEQ ID NO:45 (11.47 times increase in
comparison with WT) and SEQ ID NO:53 (8.24 times increase in
comparison with WT) was lower than SEQ ID NOS:31 and 35 (29.53
times increase) at 10 .mu.M. From these results, we thought that
maintenance of the arginine at position of WT-V had advantages.
[0156] Aspartic acid at the position of WT-VI, because SEQ ID NO:13
had a good efficiency at the low concentration (EXAMPLE 4), was
substituted by alanine or isoleucine to increase hydrophobicity. In
comparison between SEQ ID NO:31 (WT-VI:I) and SEQ ID NO:33
(WT-VI:A), translocation efficiencies of both was similarly
increased at 100 .mu.M but since increased penetrating activity of
SEQ ID NO:31 (29 times increase in comparison with WT) was far
better than SEQ ID NO:33 (3.2 times increase in comparison with WT)
at 10 .mu.M, isoleucine substitution was more effective than
alanine substitution. From these results, after this experiment,
isoleucine was introduced at the position of WT-VI of peptide
variant (from SEQ ID NOS:31, 34-36, 39).
[0157] When leucine and isoleucine at the position of WT-VII and
VIII were substituted by alanine respectively (SEQ ID NOS:14 and
15), cell penetrating activity was decreased and when both were
substituted by basic amino acids, this activity was decreased twice
(in the comparison between SEQ ID NOS:1 and 21) and when only
leucine at the position of WT-VII were substituted by glutamic
acid(E) having negative charge with strong hydrophilicity, this
activity was decreased to same degree with alanine substituent (in
the comparison between SEQ ID NOS:1 and 29) and thus it was
concluded that most effective amino acids in both positions were
leucine and isoleucine.
[0158] Serine at the position of WT-IX, when SEQ ID NO:39 (WT-IX:Y)
and SEQ ID NO:41 (WT-IX:T) substituted by each tyrosine and
threonine only at this position were compare with SEQ ID NO:31
(WT-IX:S) in cell penetrating activity, should be maintained for
the best effect. Meanwhile in all case of substitution for
tryptophan instead of methionine at the position of WT-I,
efficiency of SEQ ID NO:36 (WT-IX:S) was stronger than SEQ ID NO:40
(WT-IX:Y) and SEQ ID NO:42 (WT-IX:T) only at 10 .mu.M.
[0159] It was effective to maintain histidine(H) at the position of
WT-X. In comparison cell penetrating activity between SEQ ID NOS:1
and 2 (deletion of histidine from SEQ ID NO:1), SEQ ID NO:1 was
more effective than SEQ ID NO:2 at the concentration of 50 .mu.M
(See FIGS. 2 & 3), and when histidine was substituted by
glutamic acid (comparing SEQ ID NO:1 with SEQ ID NOS:28 and 30, See
FIGS. 7, 8 & 9), SEQ ID NO:28 and SEQ ID NO:30 were similar
with WT at 10 .mu.M and decreased 4-5 times at high
concentration.
EXAMPLE 6
Identification of Cytotoxicity of Mutant Peptides
[0160] To confirm whether cell penetrating activity of the peptides
of the present invention was due to membrane weakness as a result
of cytotoxicity, we measured cytotoxicity as follows. HeLa cells
were cultured in a 96-well plate until they were 70% grown up
before a day of the experiment. Control TAT 48-57 and the mutant
peptides at concentrations of 0, 1, 10, 100 .mu.M were treated to
DMEM supplemented with 10% FBS for 24 and 48 hours. After 2 hours
in addition of 10 .mu.l of CCK-8 to each well, absorbance at 450 nm
was measured by KC4 plate reader (FIGS. 10A, 10B, 11A, 11B, 12A and
12B). As a result of toxicity at 100 .mu.M for 24 hours,
cytotoxicity of SEQ ID NO:1, TCTP(1-10) was about 14% compared with
control, and cytotoxicities of the other peptides, TCTP-CPP#3, 7
and 8 were insignificant considering standard deviation. When
treated for 48 hours, all peptides had no cytotoxicity at 1 .mu.M
and 10 .mu.M while cytotoxicities of TAT, TCTP(1-10), TCTP-CPP#3, 7
and 8 were about 53.8, 28.3, 46.2, 8.2 and 25.6%, respectively. All
of TCTP-CPP#12-26 had no cytotoxicity at 1 .mu.M and 10 .mu.M, but
had cytotoxicity beside only TCTP-CPP#26 at 100 .mu.M. Also, all of
TCTP-CPP#27-35 had no cytotoxicity at 1 .mu.M and 10 .mu.M but had
cytotoxicity at 100 .mu.M.
Sequence CWU 1
1
84110PRTHomo sapiensMISC_FEATURE(1)..(10)1-10 amino acid residue of
TCTP 1Met Ile Ile Tyr Arg Asp Leu Ile Ser His 1 5 10 29PRTHomo
sapiensMISC_FEATURE(1)..(9)1-9 amino acid residue of TCTP 2Met Ile
Ile Tyr Arg Asp Leu Ile Ser 1 5 38PRTHomo
sapiensMISC_FEATURE(1)..(8)1-8 amino acid residue of TCTP 3Met Ile
Ile Tyr Arg Asp Leu Ile 1 5 49PRTHomo
sapiensMISC_FEATURE(1)..(9)2-10 amino acid residue of TCTP 4Ile Ile
Tyr Arg Asp Leu Ile Ser His 1 5 57PRTHomo
sapiensMISC_FEATURE(1)..(7)1-7 amino acid residue of TCTP 5Met Ile
Ile Tyr Arg Asp Leu 1 5 66PRTHomo sapiensMISC_FEATURE(1)..(6)1-6
amino acid residue of TCTP 6Met Ile Ile Tyr Arg Asp 1 5 78PRTHomo
sapiensMISC_FEATURE(1)..(8)3-10 amino acid residue of TCTP 7Ile Tyr
Arg Asp Leu Ile Ser His 1 5 89PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)M1A 8Ala Ile
Ile Tyr Arg Asp Leu Ile Ser 1 5 99PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)I2A
9Met Ala Ile Tyr Arg Asp Leu Ile Ser 1 5 109PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(9)TCTP(1-9)I3A 10Met Ile Ala Tyr Arg Asp
Leu Ile Ser 1 5 119PRTArtificial Sequencemutant peptide based on
human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)Y4A 11Met Ile Ile Ala
Arg Asp Leu Ile Ser 1 5 129PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)R5A 12Met Ile
Ile Tyr Ala Asp Leu Ile Ser 1 5 139PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)D6A
13Met Ile Ile Tyr Arg Ala Leu Ile Ser 1 5 149PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(9)TCTP(1-9)L7A 14Met Ile Ile Tyr Arg Asp
Ala Ile Ser 1 5 159PRTArtificial Sequencemutant peptide based on
human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)I8A 15Met Ile Ile Tyr
Arg Asp Leu Ala Ser 1 5 169PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(9)TCTP(1-9)S9A 16Met Ile
Ile Tyr Arg Asp Leu Ile Ala 1 5 1730DNAHomo
sapiensmisc_feature(1)..(30)DNA sequence coding 1-10 amino acid
residue of TCTP 17atgattatct accgggacct catcagccac 301827DNAHomo
sapiensmisc_feature(1)..(27)DNA sequence coding 1-9 amino acid
residue of TCTP 18atgattatct accgggacct catcagc 271910PRTHuman
immunodeficiency virusMISC_FEATURE(1)..(10)48-57 amino acid residue
of TAT 19Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10
2011PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(11)TCTP-CPP#1 20Met Ile Ile Tyr Arg Asp
Leu Ile Ser Lys Lys 1 5 10 2110PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(10)TCTP-CPP#2 21Met Ile
Ile Tyr Arg Asp Lys Lys Ser His 1 5 10 2210PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(10)TCTP-CPP#3 22Met Ile Ile Phe Arg Asp
Leu Ile Ser His 1 5 10 2310PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(10)TCTP-CPP#4 23Met Ile
Ile Ser Arg Asp Leu Ile Ser His 1 5 10 2410PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(10)TCTP-CPP#5 24Gln Ile Ile Ser Arg Asp
Leu Ile Ser His 1 5 10 2510PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(10)TCTP-CPP#6 25Cys Ile
Ile Ser Arg Asp Leu Ile Ser His 1 5 10 2612PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#7 26Met Ile Ile Tyr Arg Ala
Leu Ile Ser His Lys Lys 1 5 10 2712PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#8
27Met Ile Ile Tyr Arg Ile Ala Ala Ser His Lys Lys 1 5 10
2810PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(10)TCTP-CPP#9 28Met Ile Ile Arg Arg Asp
Leu Ile Ser Glu 1 5 10 2910PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(10)TCTP-CPP#10 29Met Ile
Ile Tyr Arg Ala Glu Ile Ser His 1 5 10 3010PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(10)TCTP-CPP#11 30Met Ile Ile Tyr Ala Arg
Arg Ala Glu Glu 1 5 10 3112PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#12 31Met Ile
Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10 3212PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#13 32Met Ile Ile Phe Arg Ala
Leu Ile Ser His Lys Lys 1 5 10 3312PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#14
33Met Ile Ile Phe Arg Ala Ala Ala Ser His Lys Lys 1 5 10
3412PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#15 34Phe Ile Ile Phe Arg Ile
Ala Ala Ser His Lys Lys 1 5 10 3512PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#16
35Leu Ile Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10
3612PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#17 36Trp Ile Ile Phe Arg Ile
Ala Ala Ser His Lys Lys 1 5 10 3712PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#18
37Trp Ile Ile Phe Arg Ala Ala Ala Ser His Lys Lys 1 5 10
3812PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#19 38Trp Ile Ile Phe Arg Ala
Leu Ile Ser His Lys Lys 1 5 10 3912PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#20
39Met Ile Ile Phe Arg Ile Ala Ala Tyr His Lys Lys 1 5 10
4012PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#21 40Trp Ile Ile Phe Arg Ile
Ala Ala Tyr His Lys Lys 1 5 10 4112PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#22
41Met Ile Ile Phe Arg Ile Ala Ala Thr His Lys Lys 1 5 10
4212PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#23 42Trp Ile Ile Phe Arg Ile
Ala Ala Thr His Lys Lys 1 5 10 4312PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#24
43Met Ile Ile Phe Lys Ile Ala Ala Ser His Lys Lys 1 5 10
4412PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#25 44Trp Ile Ile Phe Lys Ile
Ala Ala Ser His Lys Lys 1 5 10 4512PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#26
45Met Ile Ile Phe Ala Ile Ala Ala Ser His Lys Lys 1 5 10
4612PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#27 46Leu Ile Ile Phe Arg Ile
Leu Ile Ser His Lys Lys 1 5 10 4712PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#28
47Met Ile Ile Phe Arg Ile Leu Ile Ser His Lys Lys 1 5 10
4812PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#29 48Leu Ile Ile Phe Arg Ile
Leu Ile Ser His Arg Arg 1 5 10 4912PRTArtificial Sequencemutant
peptide based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#30
49Leu Ile Ile Phe Arg Ile Leu Ile Ser His His His 1 5 10
5011PRTArtificial Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(11)TCTP-CPP#31 50Leu Ile Ile Phe Arg Ile
Leu Ile Ser His Lys 1 5 10 5111PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(11)TCTP-CPP#32 51Leu Ile
Ile Phe Arg Ile Leu Ile Ser His Arg 1 5 10 5210PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(10)TCTP-CPP#33 52Leu Ile Ile Phe Arg Ile
Leu Ile Ser His 1 5 10 5312PRTArtificial Sequencemutant peptide
based on human sequenceMISC_FEATURE(1)..(12)TCTP-CPP#34 53Leu Ile
Ile Phe Ala Ile Ala Ala Ser His Lys Lys 1 5 10 5412PRTArtificial
Sequencemutant peptide based on human
sequenceMISC_FEATURE(1)..(12)TCTP-CPP#35 54Leu Ile Ile Phe Ala Ile
Leu Ile Ser His Lys Lys 1 5 10 5530DNAHomo
sapiensmisc_feature(1)..(30)DNA sequence coding TCTP-CPP#3
55atgattattt ttcgcgatct gattagccat 305636DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP CPP#7
56atgattattt atcgcgcgct gattagccat aaaaaa 365736DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#8
57atgattattt atcgcattgc ggcgagccat aaaaaa 365836DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#12
58atgattattt ttcgcattgc ggcgagccat aaaaaa 365936DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#13
59atgattattt ttcgcgcgct gattagccat aaaaaa 366036DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#14
60atgattattt ttcgcgcggc ggcgagccat aaaaaa 366136DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#15
61tttattattt ttcgcattgc ggcgagccat aaaaaa 366236DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#16
62ctgattattt ttcgcattgc ggcgagccat aaaaaa 366336DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#17
63tggattattt ttcgcattgc ggcgagccat aaaaaa 366436DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#18
64tggattattt ttcgcgcggc ggcgagccat aaaaaa 366536DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#19
65tggattattt ttcgcgcgct gattagccat aaaaaa 366636DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#20
66atgattattt ttcgcattgc ggcgtatcat aaaaaa 366736DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#21
67tggattattt ttcgcattgc ggcgtatcat aaaaaa 366836DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#22
68atgattattt ttcgcattgc ggcgacccat aaaaaa 366936DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#23
69tggattattt ttcgcattgc ggcgacccat aaaaaa 367036DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#24
70atgattattt ttaaaattgc ggcgagccat aaaaaa 367136DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#25
71tggattattt ttaaaattgc ggcgagccat aaaaaa 367236DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#26
72atgattattt ttgcgattgc ggcgagccat aaaaaa 367336DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#27
73ctgattattt ttcgcattct gattagccat aaaaaa 367436DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#28
74atgattattt ttcgcattct gattagccat aaaaaa 367536DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#29
75ctgattattt ttcgcattct gattagccat cgccgc 367636DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#30
76ctgattattt ttcgcattct gattagccat catcat 367733DNAHomo
sapiensmisc_feature(1)..(33)DNA sequence coding TCTP-CPP#31
77ctgattattt ttcgcattct gattagccat aaa 337833DNAHomo
sapiensmisc_feature(1)..(33)DNA sequence coding TCTP-CPP#32
78ctgattattt ttcgcattct gattagccat cgc 337930DNAHomo
sapiensmisc_feature(1)..(30)DNA sequence coding TCTP-CPP#33
79ctgattattt ttcgcattct gattagccat 308036DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#34
80ctgattattt ttgcgattgc ggcgagccat aaaaaa 368136DNAHomo
sapiensmisc_feature(1)..(36)DNA sequence coding TCTP-CPP#35
81ctgattattt ttgcgattct gattagccat aaaaaa 368211PRTHuman
immunodeficiency virus 82Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 10 8334PRTherpes simplex virus 1 83Asp Ala Ala Thr Ala Thr
Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1 5 10 15 Glu Arg Pro Arg
Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro 20 25 30 Val Glu
8416PRTDrosophila melanogaster 84Arg Gln Ile Lys Ile Trp Phe Gln
Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
* * * * *