U.S. patent application number 13/669414 was filed with the patent office on 2013-05-23 for peptide having cell membrane penetrating activity.
The applicant listed for this patent is Kyunglim Lee. Invention is credited to Kyunglim Lee.
Application Number | 20130129726 13/669414 |
Document ID | / |
Family ID | 38437565 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129726 |
Kind Code |
A1 |
Lee; Kyunglim |
May 23, 2013 |
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) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Lee; Kyunglim |
Seoul |
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KR |
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Family ID: |
38437565 |
Appl. No.: |
13/669414 |
Filed: |
November 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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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: |
424/134.1 ;
424/94.3; 435/188; 514/1.1; 530/350; 530/387.3; 536/23.2;
536/23.4 |
Current CPC
Class: |
A61K 47/65 20170801;
C07K 16/18 20130101; C07K 14/46 20130101; A61K 48/0008 20130101;
A61K 47/64 20170801; A61P 43/00 20180101; C07K 14/47 20130101; C07K
7/06 20130101; C07K 7/08 20130101; C07K 14/435 20130101 |
Class at
Publication: |
424/134.1 ;
530/350; 536/23.4; 536/23.2; 530/387.3; 514/1.1; 424/94.3;
435/188 |
International
Class: |
C07K 14/435 20060101
C07K014/435; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2006 |
KR |
10-2006-0016156 |
Claims
1. A transmembrane complex, comprising: a) a protein transduction
domain (PTD) that comprises all or a portion of the N-terminus of
IgE-dependent histamine-releasing factor (HRF), wherein the PTD:
has cell membrane penetrating activity; the portion contains at
least 5 amino acids; the PTD contains 5-15 amino acids; and the
N-terminus of HRF consists of the sequence MIIYRDLISH; and. b) a
target substance, whereby the transmembrane complex is not HRF.
2. A transmembrane complex of claim 1, consisting of the PTD and
the target substance.
3. A transmembrane complex of claim 1, wherein the PTD contains
8-12 amino acids.
4. The transmembrane complex of claim 1, wherein the PTD comprises
a sequence of amino acids selected from among SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 12; SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 22, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ
ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:
38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ
ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:
47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ
ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.
5. The transmembrane complex of claim 1, wherein the transmembrane
complex is a fusion protein and the fusion protein is prepared by
connecting the target substance to linker, and reacting with the
peptide having cell membrane penetrating activity to form
linkage.
6. The transmembrane complex 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.
7. A transfection kit, comprising: a) a first composition
containing a protein transduction domain (PTD) that comprises all
or a portion of the N-terminus of IgE-dependent histamine-releasing
factor (HRF), wherein the PTD: has cell membrane penetrating
activity; the portion contains at least 5 amino acids; the PTD
contains 5-15 amino acids; and the N-terminus of HRF consists of
the sequence MIIYRDLISH; and b) a second composition comprising a
target substance.
8. The transfection kit of claim 7, further comprising a binding
factor for linking the PTD to the target substance.
9. A method for delivering a target substance into a cell interior,
comprising administering a complex of claim 1 to a subject, whereby
the complex is delivered into the cell.
10. The method of claim 9, wherein the PTD comprises a sequence of
amino acids selected from among SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO:4, SEQ ID NO: 12; SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID
NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,
SEQ ID NO: 53 and SEQ ID NO: 54.
11. The method of claim 9, 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.
12. A nucleic acid molecule encoding a transmembrane complex,
comprising a sequence of nucleotides that encodes a protein
transduction domain (PTD) linked to target substance, wherein: a)
the PTD: has cell membrane penetrating activity; the portion
contains at least 5 amino acids; the PTD contains 5-15 amino acids;
and the N-terminus of HRF consists of the sequence MIIYRDLISH; and.
b) the target substance is a nucleic acid molecule encoding a
polypeptide for delivery, provided that the encoded complex is not
IgE-dependent histamine-releasing factor (HRF).
13. The nucleic acid molecule of claim 12, wherein the PTD
comprises a sequence of amino acids selected from among SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 12; SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 22,
SEQ 1D NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46,
SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID
NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.
14. The nucleic acid molecule of claim 12, wherein the target
substance is an enzyme, a regulating factor, a growth factor or an
antibody.
15. The nucleic acid molecule of claim 12, wherein the PTD is
encoded by a sequence of nucleotides selected from among one of SEQ
ID NOS: 17, 18 and 55-81.
16. A method of delivering a target substance into a cell interior,
comprising a) linking a protein transduction domain (PTD) to a
target substance by physically/chemically covalent bond or
non-covalent bond or by mediator in incorporated or fused form to
form a transmembrane complex; and b) administering the
transmembrane complex to a subject, wherein the protein
transduction domain (PTD) comprises all or a portion of the
N-terminus of IgE-dependent histamine-releasing factor (HRF),
wherein the PTD: has cell membrane penetrating activity; the
portion contains at least 5 amino acids; the PTD contains 5-15
amino acids; and the N-terminus of HRF consists of the sequence
MIIYRDLISH.
17. The method of claim 16, wherein the PTD comprises a sequence of
amino acids selected from among SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO:4, SEQ ID NO: 12; SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID
NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,
SEQ ID NO: 53 and SEQ ID NO: 54.
18. The method of claim 16, 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.
19. The transmembrane complex of claim 1 that is a fusion protein,
comprising the PTD linked to the target substance directly or via a
linker.
20. The transmembrane complex of claim 19, wherein the PTD is
linked to the target substance via a linker.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of co-pending 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, entitled "PEPTIDE HAVING CELL MEMBRANE PENETRATING
ACTIVITY," which claims priority to Korean Patent Application No.
10-2006-0016156 filed Feb. 20, 2006, to Kyunglim Lee, Moonhee Kim
and Miyoung Kim. The subject matter of each of the above-mentioned
applications is incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] 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 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.
BACKGROUND ART
[0003] Recently, various methods have been developed for delivering
macromolecules such as therapeutic drug, peptides and proteins into
cells in vitro and in vivo.
[0004] 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.
[0005] 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.
[0006] 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)
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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: HIV-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
[0011] 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).
[0012] 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.
[0013] 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 phosphoelipid bilayer of cell membrane by helix
conformation.
[0014] 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).
[0015] 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.
[0016] 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).
[0017] TCTP/HRF is considered as a histamine releasing material
interacting with basophil or mast cell and related to allergic
inflammatory response.
[0018] 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.
[0019] 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).
[0020] 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 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.
[0021] In whole structure of TCTP, N- and C-terminus get loose and
exposed and middle part forms a spherical shape.
[0022] In prediction of third structure, there are three helixes,
wherein first helix (HI) 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.
[0023] 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.
[0024] 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
[0025] 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 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.
Technical Solution
[0026] 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
[0027] 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 No.: 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 5th and 7-9th
residue(R, L, I, S)(SEQ ID Nos.: 12, 14-16) showed a little
decreased but still showed activity. Activity of
alanine-substituents of 1st-4th 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 No.: 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 No.: 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 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 Nos.: 22, 26,
27 and 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, having penetrated into a cell (either the
cytoplasm or the nucleus), become involved in the regulation of
physiological activity, have a pharmacological effect, or otherwise
maintain biological activity in the 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 forms 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.
[0072] 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(TCTP1-10)
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) atgattatttttcgcattgcggcgagccataaaaaa 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) atgattatttttcgcattgcggcgtatcataaaaaa 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) atgattatttttaaaattgcggcgagccataaaaaa 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)
ctgattatttttcgcattctgattagccataaa 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
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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, Ga14 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 vitro. Also, incorporation between DNA/RNA
binding factors and PTDs may be accomplished by chemical
interaction, physical interaction or noncovalent interaction.
[0079] 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.
[0080] 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 interacation or noncovalent interaction.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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 transmenbrane
complex into cell interior.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] The kind of the disease desired to treatment may be varied
depending on the target substance intended to administrate into
cell interior.
[0090] 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.
[0091] 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 No.: 1, 2, 22, 26, 27 or
31-54.
[0092] 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.
[0093] 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 gct.
[0094] 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.
[0095] 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
[0096] 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 on intracellular delivery in various
research fields as well as on therapeutics of specific diseases
where targeting of drugs is required high efficiently. 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.
DESCRIPTION OF DRAWINGS
[0097] 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.
[0098] FIG. 2 shows a dose dependent cellular uptake after 15
minutes of treatment of TCTP-derived peptides and 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 and FIG. 6 shows same result of FIG. 5 at the
sensitivity of 100.
[0101] FIGS. 7, 8 and 9 shows mean fluorescence intensity showing a
cellular uptake of mutant peptides of TCTP-derived peptides treated
for 2 hours at various concentrations using FACS.
[0102] FIGS. 10, 11 and 12 shows cytotoxicity of mutant peptides of
TCTP-derived peptides treated for 24 or 48 hours at a various
concentrations.
MODE FOR INVENTION
EXAMPLE 1
[0103] Mapping of PTD Using Various Deletion Forms of TCTP
[0104] 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
[0105] To overexpress each of those deletion forms of TCTP (FIGS.
1a 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
[0106] BEAS-2B cell was treated with the deletion form of TCTP at
the concentration of 15 ug/ml for 1 hour or 24 hours. Then,
supernatants and cell lysates were obtained and western blotted
with anti-TCTP antibodies (FIG. 1b).
[0107] 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 later (Lane 9). On the other hands, 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).
[0108] 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.
[0109] In addition, it was examined whether TCTP proteins of the
present invention could be transferred to cellular interior for a
short time, 5 minutes or 30 minutes. The experiment was performed
by same method as the above (FIG. 1d).
[0110] As shown in FIG. 1d, Del-C381-IRF 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.
[0111] 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
[0112] 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
[0113] TCTP-derived peptides and control peptide, TAT 48-57 were
synthesized as follow.
TABLE-US-00003 Sequence of Classification amino acid SEQ 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
[0114] N-terminus of each peptides was labeled with fluorescence
dye, rhodamine and C-terminus was protected. Peptide purity
(>95%) was determined by HPLC. Syntersis of the peptides was
requested to PEPTRON, Inc.
[0115] Negative control was a fluorescence dye, rhodamine
(Molecular Probe) used to label in all peptides.
2) Cell Culture and Incubation of Peptides
[0116] 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.
[0117] HeLa cells were cultured in 48-well plate until they were
70-80% grown up before a day of the experiment. The cells were
washed with DMEM of 37.degree. C. twice, and 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
was incubated for 15 minutes or 2 hours in an CO.sub.2 incubator at
37.degree. C.
[0118] 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).
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] So, it could be confirmed that TCTP (1-10), (1-9), (1-8) and
(2-10) peptides having cell membrane penetrating activity of the
prevent invention had superior ability than well-known PTD, TAT in
their translocation efficiency.
[0124] 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
mechan isms.
[0125] 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
[0126] 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 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).
[0127] 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 cytoplasm and nucleus of the
cell.
EXAMPLE 4
Identification of Intracellular Translocation of Peptide
Substituents
[0128] 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
[0129] Serial substituents of TCTP(1-9)(SEQ ID No.: 2) with alanine
were synthesized as follows.
TABLE-US-00004 Sequence of Classification amino acid SEQ 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
[0130] 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
[0131] 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.
[0132] HeLa cells were cultured in 48-well plate until they were
70-80% grown up before a day of the experiment. The cells were
washed with DMEM of 37.degree. C. twice, and 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 was
incubated for 15 minutes or 2 hours in an CO.sub.2 incubator at
37.degree. C.
[0133] 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).
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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
[0140] 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
[0141] 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 of Classification amino acid SEQ 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
WIIFKIAASHKK 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 LIIFRILISHK
50 TCTP-CPP#32 LIIFRILISHR 51 TCTP-CPP#33 LIIFRILISH 52 TCTP-CPP#34
LIIFAIAASHKK 53 TCTP-CPP#35 LIIFAILISHKK 54
[0142] 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
[0143] 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.
[0144] HeLa cells were cultured in 6-well plate until they were
70.about.80% grown up before a day of the experiment. The cells
were washed with DMEM of 37.degree. C. twice, and 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 was incubated for 2 hours in an CO.sub.2 incubator at
37.degree. C.
[0145] 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
[0146] 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-VII,VIII(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 analysing 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 NOS.: 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 .mu.M 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.
[0147] To analyze the role of sulfur of methionine in the position
of WT-1, 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 No.: 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 (FIG. 11). In comparison between SEQ ID
No.: 39 & 40, SEQ ID No.: 41 & 42, SEQ ID No.: 43 & 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 tM 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).
[0148] 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,
repectively. 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).
[0149] We compared substituents for basic amino acid by
substituting arginine(R) with lysine(comparison between SEQ ID No.:
31 and 43, and between SEQ ID No.: 36 and 44) or alanine(comparison
between SEQ ID No.: 31 and 45 and between SEQ ID No.: 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 No.: 31 and 35(29.53 times increase) at
10 .mu.M. From these results, we thought that maintainance of the
arginine at position of WT-V had advantages.
[0150] 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:1) and 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 No.: 31, 34-36, 39).
[0151] When leucine and isoleucine at the position of WT-VII and
VIII were substituted by alanine respectively(SEQ ID No.: 14 &
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 No.: 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 No.: 1 and 29) and thus it was
concluded that most effective amino acids in both positions were
leucine and isoleucine.
[0152] Serine at the position of WT-IX, when SEQ ID No.:
39(WT-IX:Y) and 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.
[0153] It was effective to maintain histidine(H) at the position of
WT-X. In comparison cell penetrating activity between SEQ ID No.: 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(in comparison with SEQ ID No.: 1 and 28 & 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
[0154] To confirm whether cell penetrating activity of the peptides
of present invention was due to membrane weakness as a result of
cytotoxicity, we measured cytotoxicity as follows. HeLa cells were
cultured in 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. 10, 11 and 12). 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 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 sequence 8Ala Ile Ile Tyr Arg Asp Leu Ile Ser 1 5
99PRTArtificial Sequencemutant peptide based on human sequence 9Met
Ala Ile Tyr Arg Asp Leu Ile Ser 1 5 109PRTArtificial Sequencemutant
peptide based on human sequence 10Met Ile Ala Tyr Arg Asp Leu Ile
Ser 1 5 119PRTArtificial Sequencemutant peptide based on human
sequence 11Met Ile Ile Ala Arg Asp Leu Ile Ser 1 5 129PRTArtificial
Sequencemutant peptide based on human sequence 12Met Ile Ile Tyr
Ala Asp Leu Ile Ser 1 5 139PRTArtificial Sequencemutant peptide
based on human sequence 13Met Ile Ile Tyr Arg Ala Leu Ile Ser 1 5
149PRTArtificial Sequencemutant peptide based on human sequence
14Met Ile Ile Tyr Arg Asp Ala Ile Ser 1 5 159PRTArtificial
Sequencemutant peptide based on human sequence 15Met Ile Ile Tyr
Arg Asp Leu Ala Ser 1 5 169PRTArtificial Sequencemutant peptide
based on human sequence 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
sequence 20Met Ile Ile Tyr Arg Asp Leu Ile Ser Lys Lys 1 5 10
2110PRTArtificial Sequencemutant peptide based on human sequence
21Met Ile Ile Tyr Arg Asp Lys Lys Ser His 1 5 10 2210PRTArtificial
Sequencemutant peptide based on human sequence 22Met Ile Ile Phe
Arg Asp Leu Ile Ser His 1 5 10 2310PRTArtificial Sequencemutant
peptide based on human sequence 23Met Ile Ile Ser Arg Asp Leu Ile
Ser His 1 5 10 2410PRTArtificial Sequencemutant peptide based on
human sequence 24Gln Ile Ile Ser Arg Asp Leu Ile Ser His 1 5 10
2510PRTArtificial Sequencemutant peptide based on human sequence
25Cys Ile Ile Ser Arg Asp Leu Ile Ser His 1 5 10 2612PRTArtificial
Sequencemutant peptide based on human sequence 26Met Ile Ile Tyr
Arg Ala Leu Ile Ser His Lys Lys 1 5 10 2712PRTArtificial
Sequencemutant peptide based on human sequence 27Met Ile Ile Tyr
Arg Ile Ala Ala Ser His Lys Lys 1 5 10 2810PRTArtificial
Sequencemutant peptide based on human sequence 28Met Ile Ile Arg
Arg Asp Leu Ile Ser Glu 1 5 10 2910PRTArtificial Sequencemutant
peptide based on human sequence 29Met Ile Ile Tyr Arg Ala Glu Ile
Ser His 1 5 10 3010PRTArtificial Sequencemutant peptide based on
human sequence 30Met Ile Ile Tyr Ala Arg Arg Ala Glu Glu 1 5 10
3112PRTArtificial Sequencemutant peptide based on human sequence
31Met Ile Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10
3212PRTArtificial Sequencemutant peptide based on human sequence
32Met Ile Ile Phe Arg Ala Leu Ile Ser His Lys Lys 1 5 10
3312PRTArtificial Sequencemutant peptide based on human sequence
33Met Ile Ile Phe Arg Ala Ala Ala Ser His Lys Lys 1 5 10
3412PRTArtificial Sequencemutant peptide based on human sequence
34Phe Ile Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10
3512PRTArtificial Sequencemutant peptide based on human sequence
35Leu Ile Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10
3612PRTArtificial Sequencemutant peptide based on human sequence
36Trp Ile Ile Phe Arg Ile Ala Ala Ser His Lys Lys 1 5 10
3712PRTArtificial Sequencemutant peptide based on human sequence
37Trp Ile Ile Phe Arg Ala Ala Ala Ser His Lys Lys 1 5 10
3812PRTArtificial Sequencemutant peptide based on human sequence
38Trp Ile Ile Phe Arg Ala Leu Ile Ser His Lys Lys 1 5 10
3912PRTArtificial Sequencemutant peptide based on human sequence
39Met Ile Ile Phe Arg Ile Ala Ala Tyr His Lys Lys 1 5 10
4012PRTArtificial Sequencemutant peptide based on human sequence
40Trp Ile Ile Phe Arg Ile Ala Ala Tyr His Lys Lys 1 5 10
4112PRTArtificial Sequencemutant peptide based on human sequence
41Met Ile Ile Phe Arg Ile Ala Ala Thr His Lys Lys 1 5 10
4212PRTArtificial Sequencemutant peptide based on human sequence
42Trp Ile Ile Phe Arg Ile Ala Ala Thr His Lys Lys 1 5 10
4312PRTArtificial Sequencemutant peptide based on human sequence
43Met Ile Ile Phe Lys Ile Ala Ala Ser His Lys Lys 1 5 10
4412PRTArtificial Sequencemutant peptide based on human sequence
44Trp Ile Ile Phe Lys Ile Ala Ala Ser His Lys Lys 1 5 10
4512PRTArtificial Sequencemutant peptide based on human sequence
45Met Ile Ile Phe Ala Ile Ala Ala Ser His Lys Lys 1 5 10
4612PRTArtificial Sequencemutant peptide based on human sequence
46Leu Ile Ile Phe Arg Ile Leu Ile Ser His Lys Lys 1 5 10
4712PRTArtificial Sequencemutant peptide based on human sequence
47Met Ile Ile Phe Arg Ile Leu Ile Ser His Lys Lys 1 5 10
4812PRTArtificial Sequencemutant peptide based on human sequence
48Leu Ile Ile Phe Arg Ile Leu Ile Ser His Arg Arg 1 5 10
4912PRTArtificial Sequencemutant peptide based on human sequence
49Leu Ile Ile Phe Arg Ile Leu Ile Ser His His His 1 5 10
5011PRTArtificial Sequencemutant peptide based on human sequence
50Leu Ile Ile Phe Arg Ile Leu Ile Ser His Lys 1 5 10
5111PRTArtificial Sequencemutant peptide based on human sequence
51Leu Ile Ile Phe Arg Ile Leu Ile Ser His Arg 1 5 10
5210PRTArtificial Sequencemutant peptide based on human sequence
52Leu Ile Ile Phe Arg Ile Leu Ile Ser His 1 5 10 5312PRTArtificial
Sequencemutant peptide based on human sequence 53Leu Ile Ile Phe
Ala Ile Ala Ala Ser His Lys Lys 1 5 10 5412PRTArtificial
Sequencemutant peptide based on human sequence 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
* * * * *