Patent application title: Development of Protein-Based Biotherapeutics That Induced Osteogenesis for Bone Healing Therapy: Cell-Permeable BMP2 and BMP7 Recombinant Proteins (CP-BMP2 & CP-BMP7), Polynucleotides Encoding the Same and Pro-osteogenic Compositions Comprising the Same
Inventors:
Daewoong Jo (Brentwood, TN, US)
Daewoong Jo (Brentwood, TN, US)
Eun Sin Ha (Seoul, KR)
Ji-Hye Lee (Seoul, KR)
Ji-Hye Lee (Seoul, KR)
Kyung Ae Yoon (Seoul, KR)
Bit Na Kim (Seoul, KR)
Man Young Jang (Gyeonggi-Do, KR)
IPC8 Class: AC07K1451FI
USPC Class:
530327
Class name: Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof peptides of 3 to 100 amino acid residues 11 to 14 amino acid residues in defined sequence
Publication date: 2016-03-03
Patent application number: 20160060319
Abstract:
The present invention is about direct protein delivery system for bone
morphogenetic proteins (BMPs) to enhance bone regeneration without the
need of additional delivery carrier which requires open surgery for their
implantation. The cell-permeable BMP (CP-BMP) recombinant proteins are
fused with advanced macromolecule transduction domain (aMTD) to give them
ability for cell penetration and with solubilization domain (SD) to
increase their solubility and manufacturing yield. Because existing BMP
recombinant proteins have short half-life, they have been delivered with
polymeric or inorganic vehicles for their sustained release. However,
CP-BMPs fused to combination of aMTD and SD can easily and rapidly
penetrate into the cytosol after treating on cells that likes hiding in a
shelter from wash off in body fluid, and avoid rapid degradation. For the
development of CP-BMP, BMP2 and BMP7 have been selected among various
types of BMP family due to their potent osteo-inductivity. Both of
proteins are produced in mature form (MP) as an active domain and
pro-peptide form (latency associated peptide (LAP)+MP) for prolonged
stability of proteins. In the present art, three strategic steps are used
to prove the validity of using CP-BMPs on bone regeneration and new bone
formation. First, randomly selected aMTDs and various types of SDs have
been fused to BMP proteins for determine the best structural composition
for highest solubility/yield and cell-/tissue-permeability. Next, aMTDs
are fused to BMP2 and BMP7 proteins with optimized structure to determine
the best construct for maximized cell- and tissue-permeability. Finally,
biological activity of BMP2, BMP7 and combination of BMP2 and BMP7
recombinant proteins have been evaluated to enhance in vitro osteogenic
differentiation and in vivo bone regeneration. The CP-BMPs can be applied
to repair skeletal injuries which are by bone fracture, osteogenesis
imperfecta, and bone extraction.Claims:
1. The list of amino acid sequences of BMP2 and BMP7 recombinant proteins
fused to newly invented hydrophobic cell-penetrating peptides
(CPPs)--advanced macromolecule transduction domains (aMTDs) and
solubilization domains (SDs)
2. The list of cDNA sequences of BMP2 and BMP7 recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs) and solubilization domains (SDs)
3. A list of 240 aMTD amino acid sequences according to claim 1 that satisfy all six critical factors as shown in TABLE 3.
4. Varied numbers and locations of solubilization domains (SDs) according to claim 1 that are fused to RF recombinant proteins for high solubility and yield.
5. The result of therapeutic applicability in bone regeneration with BMP2 and BMP7 recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs) and solubilization domains (SDs)
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of U.S. Provisional Application No. 62/042,493, filed on Aug. 27, 2014, in the United States Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety by reference.
TECHNICAL FIELD
[0002] The art of the invention is about cell-/tissue-permeable fusion recombinant proteins to the newly developed hydrophobic cell-penetrating peptides (CPPs) called aMTDs for enhanced bone regeneration, especially, osteoinductive fusion proteins for the recovery of bone defects caused by osteoporosis, fracture and osteoectomy. The present invention describes protocols for the production of cell-/tissue-permeable BMP2 and BMP7 recombinant proteins fused with aMTDs and solubilization domains.
BACKGROUND ART
[0003] Bone is a unique tissue that undergoes continuous remodeling throughout life and retains the potential for regeneration even in adult (1). Bone regeneration is required for bone defects caused by fracture and osteoporosis. Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor (TGF) superfamily. About 30 BMP-related proteins have been identified and can be subdivided into several groups based on their structures and functions (2). Especially, BMP2, 4 and 7 could induce chondrocyte-derived osteoprogenitor (CDOP) cell differentiation and are important in bone formation and regeneration (3-8).
[0004] BMPs are synthesized as pre-pro peptides consisting of a signal peptide (SP), latency associated peptide (LAP) and mature peptide (MP) (FIG. 1). After the synthesis, SP and LAP are later processed by enzymatic cleavage, where the C-terminal mature domain is released and secreted (9). BMPs bind to two-types of BMP receptors and signals through Smad-dependent (canonical) and Smad-independent (non-canonical) pathways (10,11). In the canonical pathway, BMP type I receptors phosphorylate receptor-regulated Smads (R-Smads). Phosphorylated R-Smads form a complex compound with common-partner Smads (Co-Smads), translocate into the nucleus and regulate the transcription of osteogenic-related genes (11).
[0005] There are four phases in the process of bone fracture repair: i) inflammatory response, ii) endochondral formation (soft callus formation and osteoblast recruitment), iii) primary bone formation (hard callus formation and mineralization), and iv) secondary bone formation (remodeling) (12-14). The bone healing process involves various associated factors including BMPs and TGF-β (15). The effect of BMPs in recombinant systems demonstrates their abilities to enhance fracture healing and skeletal defect repairs in a variety of animal models (16,17). Osteogenic potential of BMPs has allowed for their successful use as therapeutic agents for fracture healing, where enhancing bone regeneration has become general practice in spine fusion surgeries and fracture repair (18,19). The responsible genes and associated transcription factors for osteogenesis are also activated to express within a few hours of BMP treatment (20-22).
[0006] The FDA has approved the use of recombinant human BMPs (rhBMPs) including BMP2. However, rhBMPs have rapid systemic clearance and short biological half-life (7-16 min systemically and up to 8 days locally) and possible negative side-effects (ex. cancer risk) due to high dosage of BMP (23). To address these limitations, we have utilizing novel hydrophobic CPP, an advanced macromolecule transduction domain (aMTDs), to be fused to BMP proteins to have ability for cell-/tissue-permeability.
[0007] Macromolecule intracellular transduction technology (MITT) exploits the ability of aMTDs to promote bidirectional transfer of peptides across the plasma membrane. In the previous studies, previously published hydrophobic CPPs include hydrophobic region of signal sequence (HRSS)-derived short peptides called membrane-translocating motif (MTM), membrane-translocating sequence (MTS), and/or macromolecule transduction domain (MTD) in promoting proteins across the plasma membrane. In contrast to hydrophobic CPPs, cationic protein transduction domains (PTDs, e.g. those derived from HIV TAT and Antennapedia) enhance protein uptake predominately through absorptive endocytosis and macropinocytosis, which sequester significant amounts of protein into membrane-bound and endosomal compartments and limit cell-to-cell spread within the tissues.
[0008] To overcome these limitations of baseline CPPs, the aMTD sequences have been artificially composed with six critical factors, based on in-depth analysis of previously published hydrophobic CPPs, which are crucial for enhancing physiochemical properties for cell-permeability of recombinant proteins. These critical factors include amino acid length (9-13 A/a), bending potential (proline position at the middle (5', 6', 7', and 8') and at the end (12') of peptide), rigidity/flexibility (instability index (II): 40-60), structural feature (aliphatic index (AI): 180-220), hydropathy (GRAVY: 2.1-2.6), and amino acid composition (hydrophobic and aliphatic amino acids--A, V, L, I, and P) (TABLE 1). Based on these six critical factors, total of 240 aMTDs have been developed and fused to BMP for providing the cell-permeability of the recombinant fusion proteins (TABLES 2-1 to 2-6).
TABLE-US-00001 TABLE 1 [Universal Structure of Newly Develop Hydrophobic CPPs] Summarized Critical Factors of aMTD Newly Designed CPPs Critical Factor Range Bending Potential Proline presences in the middle (5', 6', 7' or 8') (Proline Position: PP) and at the end (12') of peptides Rigidity/flexibility 40-60 (Instability Index: II) Structural Feature 180-220 (Aliphatic Index: Al) Hydropathy 2.1-2.6 (Grand Average of Hydropathy GRAVY) Length 9-13 (Number of Amino Acid) Ammo acid Composition A, V, I, L, P
[0009] There has been an attempt to develop a protein-based drug with therapeutic activity; however, it had been proven difficult due to low manufacturing yield of recombinant proteins because of their low solubility in physiological condition. In addition, commercialized rhBMPs are sold in such high-cost prices, so they are not very accessible to the public. To solve this limitation, solubilization domains (SDs) have been incorporated to be fused to BMP2 and BMP7 proteins containing aMTD sequences. Consequentially, low solubility and yield had been resolved by fusing combination of aMTD/SD pair to the BMP recombinant proteins expressed in and purified from the bacteria system. Therefore, aMTD/SD-fused cell-permeable (CP)-BMP2/7 recombinant proteins have acquired much stable structure with high solubility and yield.
[0010] In the present art of invention, we hypothesize that BMP2/7 recombinant proteins fused to aMTD sequences can effectively and directly act on the bone-injured area with low concentration in a short time frame for bone regeneration. Therefore, we have developed CP-BMP2 and CP-BMP7 recombinant proteins fused to advanced macromolecule transduction domains (aMTDs) and solubilization domains to examine the effects as protein-based bio-better osteogenic agents. Development of bio-better CP-BMP2/7 will provide a great opportunity to patients for successful bone regeneration in bone-healing therapy.
SUMMARY
[0011] An aspect of the present invention relates to cell-permeable BMP2 and BMP7 recombinant proteins fused to aMTDs that are capable of macromolecule transduction into live cells for the bone healing and osteogenesis.
[0012] An aspect of the present invention relates to aMTD/SD-fused BMP2 and/or BMP7 recombinant proteins improved in solubility and manufacturing yield for clinical application.
[0013] The BMP2 and/or BMP7 proteins are described in SEQ NO: 4 and SEQ NO: 6 and they induce osteogenic differentiation in pre-osteoblasts and myoblasts.
[0014] The aMTDs are hydrophobic cell-penetrating peptides, which fully satisfy the critical factors as follows: (a) Bending potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end (12') of the sequence, (b) length: 9-13 amino acids, (c) Rigidity/Flexibility: Instability Index (II): 40-60, (d) Structural Feature: Aliphatic Index (AI): 180-220, (e) Hydropathy: GRAVY: 2.1-2.6, and (f) amino acid composition: A, V, I, L, and P.
[0015] The fusion of aMTDs to BMP2 and/or BMP7 recombinant proteins provide direct bidirectional cell-permeability across cell membrane, and it allows cell-to-cell delivery.
[0016] The combinational treatment of CP-BMP2 and CP-BMP7 synergistically enhance in vitro osteogenic differentiation and in vivo bone regeneration.
[0017] The CP-BMP2 and CP-BMP7 can be applied to bone injured area by simple injection without additional vehicles or scaffolds.
[0018] The CP-BMP2/7 recombinant proteins can be produced in both type (MP and LAP+MP: LP), and they directly uptake into cytosol within a short period of time by fusing with aMTD, which allows avoiding wash-out from the body fluid. They can be easily obtained from E. coli system with high solubility and yield by introducing customized solubilization domains. The soluble BMP LP is favorable over other types for usage because its stability could be maintained for a longer time period, which could overcome the limitations related to their short half-life. Because CP-BMP2/7 does not require any surgical procedure due to its ability of deep-tissue delivery, various administration routes could be applied and its indications could be expanded.
BRIEF DESCRIPTION OF DRAWINGS
[0019] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.
[0020] FIG. 1 shows the structural features of BMP2 and BMP7. A structural composition of BMP families is illustrated and structure design for recombinant BMPs in present invention is based on their basic structure.
[0021] FIG. 2 shows aMTD24 and aMTD123-Mediated Cell-Permeability. The cell-permeable potency of each aMTD (HM24CRA or HM123CRA) was compared to that of a Cargo A only (HCRA) (10 μM). Gray shaded area represents untreated RAW 264.7 cells (vehicle)
[0022] FIG. 3 shows aMTD24 and aMTD123-Mediated Intracellular Delivery and Localization. Fluorescence confocal laser scanning microscopy shows intracellular localization of aMTD24 or aMTD123-fused Cargo A proteins in NIH3T3 cells after incubated with 10 μM of FITC-conjugated recombinant proteins, unconjugated FITC (FITC only) or protein physiological buffer (vehicle) for 1 hour. Nomarski images are provided to show their cell morphology.
[0023] FIG. 4 shows the schematic diagram of his tagged BMP2 (MP) recombinant proteins. Design of BMP2 (MP) recombinant proteins containing histidine tag for affinity purification (MGSSHHHHHHSSLVPRGSH, white), cargo (BMP2 MP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB).
[0024] FIG. 5 shows the construction of expression for his-tagged BMP2 (MP) recombinant proteins. This figure show the agarose gel electrophoresis analysis show plasmid DNA fragments encoding BMP2 (MP) cloned into the pET28a(+) vector according to the present invention aMTD-fused BMP2(MP) and SD
[0025] FIG. 6 shows the inducible expression and purification of BMP2 (MP) recombinant proteins. Expression of BMP2 (MP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0026] FIG. 7 shows the improvement of soluble/yield of BMP2 (MP) recombinant protein with aMTD/SD-fusion. The graph compared the yield of aMTD/SD-fused BMP2 (MP) recombinant proteins with his-BMP2 (MP) Recombinant proteins lacking aMTD and SD (2M-1).
[0027] FIG. 8 shows the schematic diagram of his-tagged BMP7 (MP) recombinant proteins. Design of BMP7 (MP) recombinant proteins containing histidine tag for affinity purification (MGSSHHHHHHSSLVPRGSH, white), cargo (BMP7 MP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB).
[0028] FIG. 9 shows the construction of expression for his-tagged BMP7 (MP) recombinant proteins. These agarose gel electrophoresis analysis show plasmid DNA fragments encoding BMP7 (MP) cloned into the pET28a(+) vector according to the present invention aMTD fused BMP7 (MP) and SD
[0029] FIG. 10 shows the inducible expression and purification of BMP7 (MP) recombinant proteins. Expression of BMP7 (MP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0030] FIG. 11 shows the improvement of soluble/yield of BMP7 (MP) recombinant protein with aMTD/SD fusion. The graph compared the yield of aMTD/SD-fused BMP7 (MP) recombinant proteins with his-BMP2 (MP) Recombinant proteins lacking aMTD and SD (7M-1).
[0031] FIG. 12 shows the schematic diagram of his-tagged BMP2 (LAP+MP: LP) recombinant Proteins. Design of BMP2 (LP) recombinant proteins containing histidine tag for affinity purification (MGSSHHHHHHSSLVPRGSH, white), cargo (BMP2 LP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB).
[0032] FIG. 13 shows the construction of expression for his-tagged BMP2 (LP) recombinant proteins. These agarose gel electrophoresis analysis show plasmid DNA fragments encoding BMP2 (LAP+MP: LP) cloned into the pET28a(+) vector according to the present invention aMTD fused BMP2 (LP) and SD.
[0033] FIG. 14 shows the inducible expression and purification of BMP2 (LP) recombinant proteins. Expression of BMP2 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0034] FIG. 15 shows the structural change of BMP2 (LP) recombinant proteins. Additional designs (A, B, C) of recombinant BMP2 (LP) recombinant proteins contained histidine tag for affinity purification (white), cargo (BMP2 LP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB) or solubilization domain C (SDC).
[0035] FIG. 16 shows the construction of expression for newly designed BMP2 (LP) recombinant proteins (2L-5, 2L-6, and 2L-7). These agarose gel electrophoresis analysis show plasmid DNA fragments encoding newly designed BMP2 (LP) cloned into the pET28a(+) vector according to the present invention aMTD fused BMP2 (LP) and SD.
[0036] FIG. 17 shows the inducible expression and purification of newly designed recombinant BMP2 (LP) proteins (2L-5 and 2L-5C). Expression of BMP2 (LP) recombinant proteins (2L-5 and 2L-5C) in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE analysis which stained with Coomassie Brilliant Blue.
[0037] FIG. 18 shows the improvement of solubility/yield of recombinant BMP2 (LP) proteins (2L-5 and 2L-5C). The graph compared the yield of aMTD/SD-fused BMP2 (2L-5) recombinant proteins with His-BMP2 (LP) recombinant proteins lacking aMTD and SD (2L-1).
[0038] FIG. 19 shows the inducible expression and purification of newly designed recombinant BMP2 (LP) proteins (2L-6 and 2L-6C). Expression of BMP2 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0039] FIG. 20 shows the improvement of solubility/yield of recombinant BMP2 (LP) proteins (2L-6 and 2L-6C). The graph compared the yield of aMTD/SD-fused BMP2 (2L-6) recombinant proteins with His-BMP2 (LP) recombinant proteins lacking aMTD and SD (2L-1).
[0040] FIG. 21 shows the inducible expression and purification of newly designed recombinant BMP2 (LP) proteins (2L-7 and 2L-7C). Expression of BMP2 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0041] FIG. 22 shows the schematic diagram of His-tagged BMP7 (LP) recombinant proteins] Design of BMP7 (LP) recombinant proteins containing histidine tag for affinity purification (MGSSHHHHHHSSLVPRGSH, white), cargo (BMP7 LP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB).
[0042] FIG. 23 shows the construction of expression for His-tagged BMP7 (LP) recombinant proteins. These agarose gel electrophoresis analysis show plasmid DNA fragments encoding BMP7 (LP) cloned into the pET28a(+) vector according to the present invention aMTD fused BMP7 (LP) and SD.
[0043] FIG. 24 shows the inducible expression and purification of BMP7 (LP) recombinant proteins. Expression of BMP7 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0044] FIG. 25 shows the structural changes of BMP7 (LP) recombinant proteins. Additional designs (A, B, C) of recombinant BMP7 (LP) recombinant proteins contained histidine tag for affinity purification (white), cargo (BMP7 LAP+MP), aMTD24 (IALAAPALIVAP, black), solubilization domain A (SDA), and/or solubilization domain B (SDB) or solubilization domain C (SDC).
[0045] FIG. 26 shows the construction of expression for newly designed His-tagged BMP7 (LP) recombinant proteins] These agarose gel electrophoresis analysis show plasmid DNA fragments encoding newly designed BMP7 (LP) cloned into the pET28a(+) vector according to the present invention aMTD fused BMP7 (LP) and SD.
[0046] FIG. 27 shows the inducible expression and purification of newly designed BMP7 (LP) recombinant proteins (7L-5 and 7L-5C). Expression of BMP7 (LP) recombinant proteins (7L-5 and 7L-5C) in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0047] FIG. 28 shows the improvement of solublility/Yield of BMP7 (LP) Recombinant Proteins (7L-5 and 7L-5C)] The graph compared the yield of aMTD/SD-fused BMP7 (7L-5) recombinant proteins with His-BMP7 (LP) recombinant proteins lacking aMTD and SD (7L-1).
[0048] FIG. 29 shows the inducible expression and purification of newly designed BMP7 (LP) recombinant proteins (7L-6 and 7L-6C). Expression of BMP7 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0049] FIG. 30 shows the improvement of solubility/yield of BMP7 (LP) recombinant proteins (7L-6 and 7L-6C). The graph compared the yield of aMTD/SD-fused BMP7 (7L-6) recombinant proteins with His-BMP7 (LP) recombinant proteins lacking aMTD and SD (7L-1).
[0050] FIG. 31 shows the inducible expression and purification of newly designed recombinant BMP7 (LP) proteins (7L-7 and 7L-7C). Expression of BMP7 (LP) recombinant proteins in E. coli before (-) and after (+) induction with IPTG, and purification by Ni2+ affinity chromatography (P) were confirmed by SDS-PAGE which stained with Coomassie Brilliant Blue.
[0051] FIG. 32 shows aMTD-mediated cell-permeability of BMP2 (MP) recombinant proteins. RAW 264.7 cells (vehicle) were exposure to FITC-labeled BMP2 recombinant proteins (10 μM) compared with 4 different structures fused with (2M-2) or lacking aMTD (2M-1) and solubilization domain A (2M-3) or B (2M-4) (10 μM) for 1 hour, treated with proteinase K to remove cell associated but non-internalized proteins and analyzed by FACS. Gray shaded area represents untreated RAW 264.7 cells (vehicle) and equimolar concentration of unconjugated FITC (FITC-only, green)-treated cells were served as control.
[0052] FIG. 33 shows aMTD-mediated intracellular delivery and localization of BMP2 (MP) recombinant proteins. Fluorescence confocal laser scanning microscopy shows intracellular localization of 4 different BMP2 (MP) recombinant proteins in NIH3T3 cells after incubated with 10 μM of FITC-conjugated recombinant proteins, unconjugated FITC (FITC-only) or protein physiological buffer (vehicle) for 1 hour. Nomarski images are provided to show their cell morphology.
[0053] FIG. 34 shows aMTD-mediated cell-permeability of recombinant BMP7 (MP) recombinant proteins. RAW 264.7 cells (vehicle) were exposure to FITC-labeled BMP7 recombinant proteins (10 μM) compared with 4 different structures fused with (7M-2) or lacking aMTD (7M-1) and solubilization domain A (7M-3) or B (7M-4) (10 μM) for 1 hour, treated with proteinase K to remove cell associated but non-internalized proteins and analyzed by FACS. Gray shaded area represents untreated RAW 264.7 cells (vehicle) and equimolar concentration of unconjugated FITC (FITC-only, green)-treated cells were served as control.
[0054] FIG. 35 shows aMTD-mediated intracellular delivery and localization of BMP7 (MP) recombinant proteins. Fluorescence confocal laser scanning microscopy shows intracellular localization of 4 different BMP7 (MP) recombinant proteins in NIH3T3 cells after incubated with 10 μM of FITC-conjugated recombinant proteins, unconjugated FITC (FITC-only) or protein physiological buffer (vehicle) for 1 hour. Nomarski images are provided to show their cell morphology.
[0055] FIG. 36 shows the tissue distribution of CP-BMP2 and CP-BMP7 (MP) recombinant proteins. Cryosection of saline-perfused organs were prepared from mice 1 hour after the intraperitoneal injection of the recombinant proteins (vehicle, FITC only, FITC-2M-4C, FITC-2M-4, FITC-7M-4C or FITC-7M-4) The images from fluorescence microscopy shows distribution of CP-BMP2 and CP-BMP7 (MP) recombinant proteins in various organs.
[0056] FIG. 37 shows the schematic diagram of protocols for CP-BMP2 and CP-BMP7 recombinant proteins treatment. Schematic diagram shows that treatment schedules for osteogenic differentiation of C2C12 myoblasts and MC3T3-E1 preosteoblast which used in present invention.
[0057] FIG. 38 shows the morphological differentiation in C2C12 myoblasts with BMP2 (MP) recombinant proteins. The images of cells show the morphology of C2C12 myoblasts after treatment of BMP2 recombinant proteins with dose variation. (×100 magnification). C2C12 cells were treated with the proteins for 7 days. Proteins were freshly replaced every day. To compare the effect of CP-BMPs (2M-4), the morphology is compared with recombinant proteins lacking aMTD as well as SDs (2M-1).
[0058] FIG. 39 shows the morphological differentiation in C2C12 myoblasts with BMP7 (MP) recombinant proteins. The images of cells show the morphology of C2C12 myoblasts after treatment of BMP7 recombinant proteins with dose variation. (×100 magnification). C2C12 cells were treated with the proteins for 7 days. Proteins were freshly replaced every day. To compare the effect of CP-BMPs (7M-4), the morphology is compared with recombinant proteins lacking aMTD as well as SDs (7M-1).
[0059] FIG. 40 shows the osteogenic differentiation of myoblasts by using combinational treatment of CP-BMP2 and CP-BMP7 (MP) recombinant proteins (Protocol 1). The images of cells, which were continuously treated with vehicle or 1 μM of 2M-4 and/or 7M-4 (×100 magnification) for 7 days.
[0060] FIG. 41 shows the ALP activity of myoblasts by using combinational treatment of CP-BMP2 and CP-BMP7 (MP) recombinant proteins (Protocol 1). ALP activity of cells after 7 days of culturing with different treatment protocols.
[0061] FIG. 42 shows the steogenic differentiation of myoblasts by using combinational treatment of CP-BMP2 and CP-BMP7 (MP) recombinant proteins (Protocol 2). The images of cells, which were one-time (2 hours) treated with vehicle or 1 μM of 2M-4 and/or 7M-4 (×100 magnification) for 7 days.
[0062] FIG. 43 shows the ALP activity of myoblasts by using combinational treatment of CP-BMP2 and CP-BMP7 recombinant proteins (MP) (Protocol 2). ALP activity of cells after 7 days of culturing with different treatment protocols.
[0063] FIG. 44 shows the stimulatory effect of CP-BMP2 (MP) recombinant proteins on ALP activity in MC3T3-E1 cells. CP-BMP2 (10 μM) were continuously treated for 5 days and then measured ALP activity.
[0064] FIG. 45 shows the stimulatory effect of CP-BMP7 (MP) recombinant proteins on ALP activity in MC3T3-E1 cells. CP-BMP7 (10 μM) were continuously treated for 5 days and then measured ALP activity.
[0065] FIG. 46 shows the stimulatory effect of CP-BMP2 (MP) recombinant proteins on smad signaling in C2C12 cells. C2C12s were treated for 15 minutes with 10 μM BMP2 (MP) recombinant proteins (2M-3C, 2M-3, 2M-4C, and 2M-4) and then extrated protein in these cells. The cell lysates were analyzed for phosphorylated Smad-1/5/8 and β-actin expression.
[0066] FIG. 47 shows the stimulatory effect of CP-BMP7 (MP) recombinant proteins on smad signaling in C2C12 cells. C2C12s were treated for 15 minutes with 10 μM BMP7 (MP) recombinant proteins (7M-3C, 7M-3, 7M-4C, and 7M-4) and then extrated protein in these cells. The cell lysates were analyzed for phosphorylated Smad-1/5/8 and β-actin expression
[0067] FIG. 48 shows the osteoblastic effect of CP-BMP2 recombinant protein in calvarial injection mouse models. Hematoxylin and Eosin (H&E)-stained calvarial bone sections in diluent, 2M3C, and 2M3 recombinant protein treated groups (×400). Arrows indicate newly formed bone matrix.
[0068] FIG. 49 shows the relative activity of CP-BMP2 on new bone formation protein in calvarial injection mouse models. The graph compared the newly formed ECM thickness of aMTD/SD-fused BMP2 (2M3) and aMTD lacking SD-fused BMP2 (2M3C) recombinant proteins with protein physiological buffer (diluent).
[0069] FIG. 50 shows the osteoblastic effect of CP-BMP7 recombinant protein in calvarial injection mouse models. Hematoxylin and Eosin (H&E)-stained calvarial bone sections in diluent, 7M3C, and 7M3 recombinant protein treated groups (×400). Arrows indicate newly formed bone matrix.
[0070] FIG. 51 shows the relative activity of CP-BMP7 on new bone formation protein in calvarial injection mouse models. The graph compared the newly formed ECM thickness of aMTD/SD-fused BMP7 (7M3) and aMTD lacking SD-fused BMP7 (7M3C) recombinant proteins with protein physiological buffer (diluent).
DETAILED DESCRIPTION
1. Hypothesis of Invention
[0071] In this invention, we hypothesize that CP-BMP2 and CP-BMP7 are transmitted directly into the cell, allowing cell-to-cell delivery to avoid the rapid clearance in body fluid. Therefore, CP-BMP2/7 are capable of long term-sustainability and deep-tissue delivery. Consequentially, CP-BMP2/7 could be able to overcome the limitation of existing rhBMP2 (side effects from high dose concentration due to their short half-life and their low solubility) as protein-based bio-better osteogenic agent. To prove our hypothesis, we have developed CP-BMP2/7 recombinant proteins fused with novel hydrophobic CPPs called aMTDs to obtain cell-/tissue-permeability, and additionally fused with solubilization domains to increase their solubility and yield in the physiological condition. These CP-BMP2/7 recombinant proteins have shown to greatly improve their solubility and cell-permeability.
[0072] Through this invention, we expect that exogenously administered BMP2 and BMP7 proteins can enhance bone formation during the healing of bone fracture or steady-state condition. CP-BMP2/7 can be effectively and rapidly delivered into the neighboring cells and tissues nearby the injured site, which makes the recombinant proteins to be relatively free from rapid degradation and clearance issues compared to other recombinant human BMPs (rhBMPs). Therefore, CP-BMP2/7 can overcome previously indicated limitations and provide various administration routes for bone healing therapy at relatively low cost.
2. Determination of Optimized Structure of BMP2 and BMP7 Recombinant Proteins
[0073] 2-1. Novel Hydrophobic Cell-Penetrating Peptide (CPP)--Advanced Macromolecule Transduction Domain (aMTD)
2-1-1. Analysis of Hydrophobic CPPs
[0074] Many proteins having a basic peptide sequence that bind heparin sulfate proteoglycans, including cationic cell-penetrating peptides, such as HIV-1 Tat-derived protein transduction domain (PTD), enter cells by caveolin-dependent and independent endocytosis. The bulk uptake often exceeds and therefore masks a smaller, a biologically active component that enters the cytoplasm either by escaping the vesicular compartment or by alternative routes, e.g. one involving higher affinity (but less abundant) receptors (24). Vesicular sequestration of basic proteins typically limits tissue penetration and bioavailability, thus hampering efforts to develop protein-based therapeutics.
[0075] In contrast to cationic CPPs, hydrophobic CPPs such as MTD sequences appear to penetrate the plasma membrane directly after inserting into the membranes. In action mechanism, MTD-facilitated uptake of larger proteins is sensitive to low temperature, does not require microtubule function (no endocytosis) or utilize ATP (no energy source), and intracellular accumulation requires an intact plasma membrane. In principle, therefore, crucial features such as cell-to-cell transfer and tissue penetration mediated by hydrophobic CPP such as MTD make these peptide sequences to deliver therapeutic cargo proteins in living cells and animals to treat various lethal disorders including cancer.
[0076] To address the limitation of previously developed hydrophobic CPPs, novel sequences have been developed. To design new hydrophobic CPPs for intracellular delivery of cargo proteins such as BMPs, identification of optimal common sequence and/or homologous structural determinants, namely critical factors (CFs), had been crucial. To do it, the physicochemical characteristics of previously published hydrophobic CPPs were analyzed. To keep the similar mechanism on cellular uptake, all CPPs analyzed were hydrophobic region of signal peptide (HRSP)-derived CPPs (e.g. membrane translocating sequence: MTS and macromolecule transduction domain: MTD) as explained previously.
(1) Basic Characteristics of CPPs Sequence
[0077] These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzed for their 11 different characteristics--sequence, amino acid length, molecular weight, pl value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition, and secondary structure of the sequences. Two peptide/protein analysis programs were used (ExPasy: http://web.expasy.org/protparam/, SoSui: http://harrier.nagaharna-i-bia.ac.jp/sosui/sosui_submit.html) to determine various indexes, structural features of the peptide sequences and to design new sequence. The following factors have been considered important. Average length, molecular weight and pl value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively.
(2) Bending Potential (Proline Position: PP)
[0078] Bending potential (bending or no-bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences the protein architecture, which is often found in the bends of folded peptide/protein chain. Eleven out of 17 were determined as `bending` peptide, which meant that proline have be present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate through the plasma membrane in a "bent" configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.
(3) Rigidity/Flexibility (Instability Index: II)
[0079] Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9-79.1), but average value was 40.1±21.9, which suggested that the peptide should be somehow flexible, but not too rigid or flexible.
(1) Hydropathy (Grand Average of Hydropathy: GRAVY) and Structural Feature (Aliphatic Index: AI)
[0080] Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic--that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively.
(2) Secondary Structure (α-Helix)
[0081] As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a "bent" configuration with hydrophobic sequences adopting an α-helical conformation. In addition, our analysis strongly indicated that bending potential was crucial. Therefore, structural analysis of the peptides conducted to determine whether the sequence was to form helix or not. Nine peptides were helix and 8 were not. It seems to suggest that helix structure may not be required, but favored for membrane penetration.
(3) Determination of Critical Factors (CFs)
[0082] In the 11 characteristics analyzed, the following 6 are selected namely "Critical Factors (CFs)" for the development of new hydrophobic CPPs--advanced MTDs: i) amino acid length, ii) bending potential (proline presence and location), iii) rigidity/flexibility (instability index: II), iv) structural feature (aliphatic index: AI), v) hydropathy (GRAVY) and vi) amino acid composition/residue structure (hydrophobic and aliphatic A/a).
2-1-2. Analysis of Selected Hydrophobic CPPs to Optimize `Critical Factors`
[0083] Since the analyzed data of the 17 different hydrophobic CPPs (analysis A) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus-features, additional analysis B and C was also conducted to optimize the critical factors for better design of improved CPPs--aMTDs.
[0084] In analysis B, 8 CPPs were used with each cargo in vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/flexibility was 41±15, but removing one [MTD85: rigid, with minimal (II: 9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially better. All other characteristics of the 8 CPPs were similar to the analysis A including structural feature and hydropathy.
[0085] To optimize the `common range and/or consensus feature of critical factor` for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the `Critical Factors` determined in the analysis A, B was correct to improve the current problems of hydrophobic CPPs--protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.
[0086] The peptides which did not have a bending potential, rigid or too flexible sequences (too low or too high instability index), or too low or too high hydrophobic CPP were unselected, but secondary structure was not considered because helix structure of sequence was not required. 8 selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed. Common amino acid length is 12 (11.6±3.0). Proline should be presence in the middle of and/or the end of sequence. Rigidity/flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs (TABLE 1).
[0087] 1. Amino Acid Length: 9-13
[0088] 2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5' to 8' amino acid) and at the end of sequence
[0089] 3. Rigidity/Flexibility (Instability Index: II): 40-60
[0090] 4. Structural Feature (Aliphatic Index: AI): 180-220
[0091] 5. Hydropathy (GRAVY): 2.1-2.6
[0092] 6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids--A, V, L, I and P
2-1-3. Determination of Critical Factors for Development of aMTDs
[0093] To confirm the validity of 6 critical factors providing the optimized cell-/tissue-permeability, all 240 aMTD sequences have been designed and developed based on these critical factors (TABLES 2-1 to 2-6). All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12 A/a-length peptides) are practically confirmed by their quantitative and visual cell-permeability. To determine the cell-permeability of aMTDs, rPeptides, that do not satisfy one or more critical factors have also been designed and tested. Relative cell-permeability of 240 aMTDs to the negative control (random peptide (rP) 38, hydrophilic & non-aliphatic 12 A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. Moreover, compared to the reference CPPs (MTS/MTM1 and MTD), novel 240 aMTDs showed averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively. As a result, the association of cell-permeability of the peptides and critical factors was vivify displayed. Based on the result from the newly designed and tested novel 240 aMTDs, the empirically optimized critical factors (CFs) are provided below.
[0094] 1. Amino Acid Length: 12
[0095] 2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5' to 8' amino acid) and at the end of sequence
[0096] 3. Rigidity/Flexibility (Instability Index: II): 41.3-57.3
[0097] 4. Structural Feature (Aliphatic Index: AI): 187.5-220.0
[0098] 5. Hydropathy (GRAVY): 2.2-2.6
[0099] 6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids--A, V, L, I and P
TABLE-US-00002 TABLE 2-1 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 1-46)] ##STR00001##
TABLE-US-00003 TABLE 2-2 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 47-92)] ##STR00002##
TABLE-US-00004 TABLE 2-3 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 93-138)] ##STR00003##
TABLE-US-00005 TABLE 2-4 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 139-184)] ##STR00004##
TABLE-US-00006 TABLE 2-5 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 185-230)] ##STR00005##
TABLE-US-00007 TABLE 2-6 [Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are Considered and Satisfied (aMTD 231-240)] ##STR00006##
[0100] These examined factors are within the range that we have set for our critical factors; therefore, we were able to confirm that the a MTDs that satisfy these critical factors have much higher cell-permeability (TABLE 3) and intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.
TABLE-US-00008 TABLE 3 [Summarized Critical Factors of aMTD after In-Depth Analysis of Experimental Results] Summarized Critical Factors of aMTD Analysis of Experimental Results Critical Factor Range Bending Potential Proline presences in the middle (5', 6', 7' or 8') (Proline Position: PP) and at the end (12') of peptides Rigidity/Flexibility 41.3-57.3 (Instability Index: II) Structural Feature 187.5-220.0 (Aliphatic Index: Al) Hydropathy 2.2-2.6 (Grand Average of Hydropathy GRAVY) Length 12 (Number of Amino Acid) Amino acid Composition A, V, I, L, P
2-2. aMTD24 and aMTDp123 were Selected for Cell-Permeability of BMP Recombinant Proteins
[0101] To develop CP-BMPs, aMTD24 and p123 were randomly selected and fused to BMP recombinant proteins to provide cell permeability. Characteristics of aMTD24 and aMTDp123 are provided in TABLE 4, and the information demonstrated that they are completely satisfying to `critical factors`. The cell permeability of selected aMTDs are evaluated by FACS analysis as shown in FIG. 2. The fusion of aMTD24 or aMTDp123 to Cargo A protein showed much enhanced cell penetration than that of Cargo A protein lacking aMTDs which resulted in shifting of peak to the right. Next, the intracellular distribution of aMTD fused proteins is visualized by using FITC-conjugated proteins. As shown in FIG. 3, there are no fluorescence in vehicle, FITC only, HCRA (Cargo A protein lacking aMTD), and HrP38CRA (Cargo A protein fused with random peptide 38). However, aMTD24 or aMTDp123 fused proteins are observed in cytosol of cells. These results demonstrate that the selected aMTD24 and aMTDp123 possess sufficient cell-permeability to penetrate through the cell membrane.
TABLE-US-00009 TABLE 4 [Characteristics of aMTD24 and aMTD p123] Bending potential Rigidity/ Sturctural aMTD A/a Proline Position Flexibility Feature Hydropathy ID Sequence Length 5' 6' 7' 8' 12' Number (II) (Al) (GRAVY) 24 IALAAPALIVAP 12 0 1 0 0 1 2 50.2 195.8 2.2 p123 PAAIIVPAALLA 12 0 1 0 0 1 2 50.2 195.8 2.2
2-3. Solubilization Domains were Fused for Stable Structure of CP-BMP2 (MP) Recombinant Proteins
[0102] We designed 4 different types of recombinant proteins with or lacking the aMTD24 and solubilization domains (SDs) for BMP2 mature protein (MP). Protein structures were labeled as follows: 1) 2M-1, a BMP2 MP only, 2) 2M-2, a BMP2 MP fused with aMTD, 3) 2M-3, a BMP2 MP fused with aMTD and solubilization domain A (SDA) and 4) 2M-4, a BMP2 MP fused with aMTD and solubilization domain B (SDB) (FIG. 4). Control proteins lacking aMTD were designed separately, as 2M-3C (a BMP2 MP fused with SDA lacking aMTD) and 2M-4C (a BMP2 MP fused with SDB lacking aMTD). The expression vectors were successfully constructed (FIG. 5) and cloned for protein expression and purification. FIG. 6 shows that each type of BMP2 MP recombinant proteins were successfully expressed and purified from E. coli. Although, 2M-1 and 2M-2 showed insoluble features, 2M-3 and 2M-4 showed significantly improved solubility due to fused SDs at C-terminal of recombinant proteins. Relative protein yield was significantly increased by fusing SDA (5-folds) or SDB (10-folds) compared to control protein (2M-1) (FIG. 7).
2-4. Solubilization Domains were Fused for Stable Structure of CP-BMP7 (MP) Recombinant Proteins
[0103] We designed 4 different types of recombinant proteins with or lacking the aMTD24 and solubilization domains (SDs) for BMP7 mature protein (MP). Protein structures were labeled as follows: i) 7M-1, a BMP7 MP only, ii) 7M-2, a BMP7 MP fused with aMTD, iii) 7M-3, a BMP7 MP fused with aMTD and solubilization domain A (SDA) and iv) 7M-4, a BMP7 MP fused with aMTD and solubilization domain B (SDB) (FIG. 8). Control proteins lacking aMTD were designed separately, as 7M-3C (a BMP7 MP fused with SDA lacking aMTD) 7M-4C (a BMP7 MP fused with SDB lacking aMTD). The expression vectors were successfully constructed (FIG. 9) and cloned for protein expression and purification. FIG. 10 show that each type of BMP7 MP recombinant proteins were successfully expressed and purified from E. coli. Although, 7M-1 and 7M-2 showed insoluble features, 7M-3 and 7M-4 showed significantly improved solubility due to fused SDs at C-terminal of recombinant proteins. Relative protein yield was significantly increased by fusing SDA (80-folds) or SDB (100-folds) compared to control protein (7M-1) (FIG. 11).
2-5. Solubilization Domains were Fused for Stable Structure of CP-BMP2 (LAP+MP) Recombinant Proteins
[0104] Because of the BMP proteins are composed of 3 parts (signal sequence, latency associated peptide (LAP) and mature peptide (MP)), we also designed 4 new types of recombinant proteins by replacing BMP MP with BMP LAP+MP (LP) protein (FIG. 12). The expression vectors for each BMP2 LP proteins were successfully constructed and cloned for protein expression and purification (FIG. 13). BMP2 LP recombinant proteins were successfully induced by adding IPTG and purified. However, they showed very limited solubility and yield in all types of structure, despite the fact that BMP2 LP proteins were fused with SDs to improve their solubility (FIG. 14).
[0105] In order to solve the problem with low solubility and yields, additional 3 sets of structures for BMP2 LP recombinant proteins were designed as shown in FIG. 15. aMTDp123 was fused to each type of BMP2 LP recombinant proteins to give ability for cell penetration. Recombinant proteins in A (2L-5 and 2L-5C) are fused with SDBs on both N-terminal and C-terminal with or lacking aMTD. Recombinant proteins in B (2L-6 and 2L-6C) are combinational fusion of SDA on N-terminal and SDB on C-terminal of proteins with or lacking aMTD. Proteins in C (2L-7 and 2L-7C) are fused with SDC on N-terminal of proteins with or lacking aMTD. The expression vectors for newly designed BMP2 LP proteins were successfully constructed and cloned for protein expression and purification (FIG. 16). 2L-5 and 2L-5C proteins were successfully expressed and purified with significantly improved solubility (FIG. 17). The relative yield also significantly increased in 2L-5 and 2L-5C protein (48 and 90 folds, respectively) compared to 2L-1 protein (FIG. 18). 2L-6 and 2L-6C proteins were also successfully purified with great solubility and they showed 54 folds and 47 folds increase of yield, respectively (FIGS. 19 and 20). In contrast to BMP2 LP 5 and 6 proteins, 2L 7 and 2L-7C proteins were induced by addition of IPTG and purified, but they showed very limited solubility and yield (FIG. 21). These results demonstrate that the combinational fusion of SDA and/or SDB to BMP2 LP proteins significantly improve their solubility, while SDC fused BMP2 LP proteins showed indifference.
2-6. Solubilization Domains were Fused for Stable Structure of CP-BMP7 (LAP+MP) Recombinant Proteins
[0106] Recombinant BMP7 LP proteins were designed in 4 different types same as BMP2 LP proteins (FIG. 22). aMTDp123 was fused to each type of BMP2 LP recombinant proteins to give ability for cell penetration. Four different expression vectors for BMP7 LP proteins were successfully constructed and cloned for protein expression and purification (FIG. 23). Although, BMP7 LP recombinant proteins were successfully induced by adding IPTG and purified, we failed to obtain soluble proteins in all types of BMP7 LP proteins (FIG. 24).
[0107] In order to solve the problem with low solubility and yields, additional 3 sets of structures for BMP7 LP recombinant proteins were designed as shown in FIG. 25. The expression vectors for newly designed BMP7 LP proteins were successfully constructed and cloned for protein expression and purification (FIG. 26). The 7L-5 and 7L-5C proteins were successfully expressed and purified with significantly improved solubility (FIG. 27). The 7L-5 and 7L-5C protein showed significant increase of yields (78- and 68-folds, respectively) relative to 7L-1 protein (FIG. 28). 7L-6 and 7L-6C proteins also successfully purified with great solubility and they showed 57-folds and 34-folds increase of yield compared to 7L-1, respectively (FIGS. 29 and 30). Similar with 2L-7 and 2L-7C proteins, 7L-7 and 7L-7C were induced by addition of IPTG and purified, but solubility and yield were not improved by fusing with SDC (FIG. 31).
TABLE-US-00010 TABLE 5 [Characteristics of Solubilization Domain] Protein Instability SD Genhank ID Origin (kDa) pl Index (II) GRAVY A CP000113.1 Bacteria 23 4.6 48.1 -0.1 B BC086945.1 Pansy 11 4.9 43.2 -0.9 C CP012127.1 Human 12 5.8 30.7 -0.1 D CP012127.1 Bacteria 23 5.9 26.3 -0.1 E CP011550.1 Human 11 5.3 44.4 -0.9 F NG_034970 Human 34 7.1 56.1 -0.2
3. Determination of Cell-/Tissue-Permeability of Each Recombinant Protein
[0108] 3-1. aMTD/SD-Fused CP-BMP2/7 Show Great Cell-Permeability.
[0109] Because we first secured full set of purified recombinant BMP2 and BMP7 MP (mature peptide) proteins, BMP2 MP and BMP7 MP were used for further investigations including in vitro/in vivo permeability and biological activity tests.
[0110] Cell-permeability of BMP2 MP and BMP7 MP in vitro was evaluated in Raw 264.1 cells after 1 hour of protein treatment (FIGS. 32 and 34). BMP2 and BMP7 proteins lacking aMTD (2M-1 and 7M-1) showed limited cell penetration. An employment of aMTD increased cell-permeability in both BMP2 and BMP7 (2M-2, 7M-2). In addition,
[0111] In addition, SDs synergistically increased the cell-permeability (2M-3, 4 and 7M-3, 4). Protein type 4, composed with aMTD and SDB (2M-4, 7M-4) showed the highest cell-permeability.
[0112] The results perfectively matched with the result from confocal microscopy (FIGS. 33 and 35). Highest signal intensity was observed in protein type 4 (2M-4, 7M-4), which indicated a successful uptake of proteins into the cells. These results showed that the aMTD enabled the proteins to enter the cells within short time (1 hour) and improved the solubility of proteins that positively affect cell-permeability.
3-2. MTD/SD-Fused CP-BMP2/7 Recombinant Proteins Gain In Vivo Tissue-Permeability.
[0113] Next, we determined in vivo tissue-permeability of recombinant CP-BMP2 and CP-BMP7 proteins after 2 hours of intraperitoneal injection of FITC-labeled proteins (FIG. 36). There was no signal of fluorescence in all tested organs of vehicle and FITC only after the injection. We used 2M-4C and 7M-4C as control for protein type 4 (2M-4 and 7M-4), which the cargo proteins are fused with SDB but not aMTD. The control protein type 4 (2M-4C and 7M-4C) showed limited tissue permeability in some organs (heart, lung, liver and kidney) and were not detected in the brain and spleen. In contrast, aMTD24 fused to BMP2 and BMP7 proteins enhanced the systemic delivery of BMP2 and BMP7 in some tissues (heart, lung, liver and kidney). However, the fusion of aMTD to BMP proteins has not significantly improved the systemic delivery of BMP2 and BMP7 in the brain and spleen.
4. Determination of In Vitro Biological Activity of CP-BMP2/7 Recombinant Proteins
4-1. CP-BMP2/7 Recombinant Proteins Enhance Osteogenic Differentiation of C2C12 Myoblasts in Dose-Dependent Manner.
[0114] To examine the effect of CP-BMP2 MP and CP-BMP7 MP on the osteogenic differentiation of C2C12 myoblasts, we have designed two protocols with varied exposure times of CP-BMPs (FIG. 37). C2C12 myoblasts are known to differentiate into myotubes under the starvation condition (<2% of FBS or horse serum in media), and the treatment of BMPs suppress myogenesis and lead to osteogenesis. Both protocols are set in a serum free condition during 2 hours of protein treatment for efficient permeability of CP-proteins. In Protocol 1, CP-BMPs are treated in serum free condition for 2 hours and continuously exposed in 2% FBS media for 7 days. In protocol 2, CP-BMPs are washed out with PBS after 2 hours of exposure and then incubated for 7 days under 2% FBS media without any additional treatment of CP-BMPs.
[0115] The effect of CP-BMP2 MP and CP-BMP7 MP on osteogenic differentiation of C2C12 myoblasts is determined by treating BMPs in various doses (FIGS. 38 and 39). We have compared the effect of CP-BMPs using 2M-1/7M-1 (cargo lacking aMTD) and 2M-4/7M-4 (cargo with aMTD and SDB), which has shown greatest cell-/tissue-permeability. The inhibitory effects on myotube formation of C2C12 cells are not shown at the low concentrations (0.1 and 0.5 μM) of 2M-1, 7M-1, 2M-4 and 7M-4. However, treatment of 2M-1 or 7M-1 at 1 μM significantly inhibited the myotube formation, which manifests the transition of lineage differentiation from myogenic to osteogenic. Highest concentration 5 μM of 2M-1 has shown weak cytotoxicity, while same dose of 7M-1 has shown strong inhibition of myotubes formation without any cytotoxic effect. Unlike what has been previously expected, 2M-4 and 7M-4 did not affect and differentiation of C2C12 cells even at the high doses (1 and 5 μM). Therefore, we have selected 1 μM of BMP2 MP and BMP7 MP as the effective concentration for further experiments.
4-2. Combinational Treatment of CP-BMP2 and CP-BMP7 Provide Synergistic Effect on Osteogenesis of C2C12 Myoblasts.
[0116] Synergistic effect of CP-BMP2 and CP-BMP7 on osteogenic differentiation of C2C12 myoblasts was evaluated with two different protocols as described in FIG. 37. When the cells were continuously exposed to CP-BMP2 and/or CP-BMP7 recombinant proteins for 7 days, vehicle showed lots of large extended myotubes with aligned direction. In addition, 2M-4 and 7M-4 also showed very similar morphology of myotubes in vehicle group. On the other hand, significant prevention of myotubes formation was observed in the combinational treatment of 2M-4 and 7M-4 (FIG. 40). In ALP assay, it showed superior ALP activity following the combinational treatment of 2M-4 and 7M-4 compared to others (FIG. 41).
[0117] Next, cells were exposed to the proteins for only first 2 hours and then incubated without proteins for additional 7 days. Cells were mainly differentiated into myotubes without any treatment of BMPs (vehicle), and the same result was also observed when the cells were exposed to 2M-4 and 7M-4 for a short period of time. Although the cells were treated with the proteins for only 2 hours, a significant inhibitory effect on myotubes formation was shown in combinational treatment of 2M-4 and 7M-4 (FIG. 42). While the synergistic effect of BMP2 MP and BMP7 MP combinational treatment was observed in ALP activity, the activity level was significantly lower than that of BMP2 MP proteins (FIG. 43). These results showed that sufficient exposure time of CP-BMPs is required for effective osteogenic differentiation. Together, combinational treatment of CP-BMP2 and CP-BMP7 synergistically induced the osteogenic differentiation of C2C12 cells.
4-3. Combinational Treatment of CP-BMP2 and CP-BMP7 Provide Synergistic Effect on Osteogenesis of MC3T3-E1 Pre-Osteoblasts.
[0118] MC3T3-E1, pre-osteoblast also used to evaluate the effect of CP-BMP2 MP and CP-BMP7 MP on osteogenic differentiation. To determine the osteogenic differentiation, each protein was treated on MC3T3-E1 cells every day and ALP activity was measured at 5 days after protein treatment. In the case of CP-BMP2 MP, the similar level of ALP activity compared to vehicle was observed in 2M-3C, 2M-4C, and 2M-4. However, 2M-3 resulted in 3.5 fold increase of ALP activity (FIG. 44). On the other hand, 7M-3 and 7M-4 that were fused with aMTD showed approximately 3 folds greater ALP activity compared to their control proteins lacking aMTD (7M-3C and 7M-4C) (FIG. 45). These results revealed that the incorporation of aMTDs to BMP2 protein with SDA (2M-3) can enhance the osteogenic differentiation of pre-osteoblasts. In addition, the incorporation of aMTDs to BMP2 protein with SDA or SDB can facilitate osteogenic differentiation of pre-osteoblasts.
4-4. CP-BMP2 and CP-BMP7 Recombinant Proteins Activate Smad-Mediated Signaling Pathway.
[0119] To confirm biological activity of CP-BMPs in C2C12 cells, we have investigated the activation of Smad-signaling. For starvation of cells, confluent C2C12 cells were incubated with serum free DMEM media, and then 10 μM of 4 different CP-BMP2 MP and CP-BMP7 MP proteins were separately treated for 15 minutes.
[0120] The treatment of 2M-3 induced strong phosphorylation of Smad 1/5/8, whereas other CP-BMP2 MP proteins (2M-3C, 2M-4C, and 2M-4) did not induce Smad-signaling in C2C12 cells (FIG. 46). On the other hand, treatment of 7M-3C showed strongest expression of pSmad1/5/8 compare to the others. Next, 7M-3 showed relatively strong, comparatively stronger than 7M-4C, expression of pSmad1/5/8. However, 7M-4 did not induce phosphorylation of Smad 1/5/8 in C2C12 cells (FIG. 47). These results demonstrated that the 2M-3 can be selected as an optimized structure for CP-BMP2 MP which shows sufficient biological activity. However, optimized structure for CP-BMP7 MP should be further examined by changing with 10 different aMTDs.
5. CP-BMP2/7 Recombinant Proteins Enhance New Bone Formation in Calvarial Injection Assay.
[0121] To investigate the effect of CP-BMP2 and CP-BMP7 on in vivo new bone formation of calvaria, each CP-BMP was locally injected to their calvaria by subcutaneous injection as described in example section. After 4 weeks, new bone formation was determined by using H&E staining. As shown in FIG. 48, only few lining cells were observed on the surface of calvarial bone tissue in diluent treated group. In 2M3C treated group, the BMP2 protein without aMTD, showed increase of extra cellular matrix (ECM) formation on the surface of calvaria tissue, which indicated that the immature bone matrix formation. On the other hands, the significant increase of ECM formation was observed in 2M-3 treated group, the BMP2 protein fused with aMTD. The new bone formation was quantified by measuring their newly formed ECM thickness (FIG. 49). Although the 2M-3C treated group showed more than 5 folds greater relative activity, 2M-3 treated group showed more than 20 folds greater relative activity which compare to diluent treated group. The result of CP-BMP7 was very similar to the result of CP-BMP2 (FIG. 50). Only few cells were lining on the calvaria in diluent treated group. However, the injection of 7M-3C, the BMP7 protein without aMTD, resulted in the increase of the immature bone matrix formation. On the other hands, 7M-3 treated group, the BMP7 protein fused with aMTD, showed increased dense bone matrix formation which composed with lots of cells. In quantification results, 7M-3C showed 5 folds higher activity while 7M-3 showed more than 25 folds higher activity relative to diluent treated group. These results showed that the fusion of aMTD to BMP2 or BMP7 recombinant proteins resulted in great increase of their bioactivity such as new bone formation.
EXAMPLES
[0122] The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the invention.
Example 1
Design of Novel Hydrophobic CPPs--aMTDs for Development of CP-BMP2/7
[0123] As mentioned above, H-regions of signal peptides (HRSP)-derived CPPs (MTM, MTS and MTD) do not have a common sequence, sequence motif and/or common-structural homologous feature. In this invention, the aim is to develop CP-BMP2/7 by adopting novel hydrophobic CPPs formatted in the common sequence- and structural-motif, which satisfy newly determined `critical factors` to have `common function`, namely, to facilitate protein translocation across the membrane with similar mechanism to the analyzed CPPs. It is also suggested that the length of 12 amino acids; and the bending potential is provided with the presence of proline in the middle of sequence (at 5', 6', 7' or 8' amino acid) for peptide bending and at the end of peptide (at 12') for recombinant protein bending. Rigidity/flexibility of aMTDs is around II 50, and the structural features are described in TABLE 9 in detail. The analysis of selected published CPPs is based on the critical factors, the novel hydrophobic CPPs--aMTDs--are designed for the development of CP-BMP2/7 proteins; and their critical factors are also analyzed and compared to the selected CPPs (TABLE 9).
TABLE-US-00011 TABLE 9 [PCR Primers for His-tagged BMP7 (MP) Proteins Fused to Different 17 Kinds of aMTDs] A/a 5' Primer 3' Primer ID Sequence (5' → 3') (5' → 3') 1 AAALAPVVLALP ATTTATCATATGGCG CGCGTCGAC GCGGCGCTGGCGCCG TTACCTCGG GTGGTGCTGGCGCTG CTGCACCGG CCGACGCCCAAGAAC CACGGAGAT CAGGAAGCC GAC 2 AALLVPAAVLAP ATTTATCATATGGCG GCGCTGCTGGTGCCG GCGGCGGTGCTGGCG CCGACGCCCAAGAAC CAGGAAGCC 3 VAALPVLLAALP ATTTATCATATGGTG GCGGCGCTGCCGGTG CTGCTGGCGGCGCTG CCGACGCCCAAGAAC CAGGAAGCC 4 AAAVVPVLLVAP ATTTATCATATGGCG GCGGCGGTGGTGCCG GTGCTGCTGGTGGCG CCGACGCCCAAGAAC CAGGAAGCC 5 IVAIAVPALVAP ATTTATCATATGATT GTGGCGATTGCGGTG CCGGCGCTGGTGGCG CCGACGCCCAAGAAC CAGGAAGCC 6 AAALVIPAAILP ATTTATCATATGGCG GCGGCGCTGGTGATT CCGGCGGCGATTCTG CCGACGCCCAAGAAC CAGGAAGCC 7 ALAALVPAVLVP ATTTATCATATGGCG CTGGCGGCGCTGGTG CCGGCGGTGCTGGTG CCGACGCCCAAGAAC CAGGAAGCC 8 VLVALAAPVIAP ATTTATCATATGGTG CTGGTGGCGCTGGCG GCGCCGGTGATTGCG CCGACGCCCAAGAAC CAGGAAGCC 9 IVAVALPALAVP ATTTATCATATGATT GTGGCGGTGGCGCTG CCGGCGCTGGCGGTG CCGACGCCCAAGAAC CAGGAAGCC 10 IAVALPALIAAP ATTTATCATATGATT GCGGTGGCGCTGCCG GCGCTGATTGCGGCG CCGACGCCCAAGAAC CAGGAAGCC 11 ALAVIVVPALAP ATTTATCATATGGCG CTGGCGGTGATTGTG GTGCCGGCGCTGGCG CCGACGCCCAAGAAC CAGGAAGCC 12 AVVIALPAVVAP ATTTATCATATGGCG GTGGTGATTGCGCTG CCGGCGGTGGTGGCG CCGACGCCCAAGAAC CAGGAAGCC 13 LVAIVVLPAVAP ATTTATCATATGCTG GTGGCGATTGTGGTG CTGCCGGCGGTGGCG CCGACGCCCAAGAAC CAGGAAGCC 14 AIAIAIVPVALP ATTTATCATATGGCG ATTGCGATTGCGATT GTGCCGGTGGCGCTG CCGACGCCCAAGAAC CAGGAAGCC 15 VAAAIALPAIVP ATTTATCATATGGTG GCGGCGGCGATTGCG CTGCCGGCGATTGTG CCGACGCCCAAGAAC CAGGAAGCC 16 AVIVPVAIIAAP ATTTATCATATGGCG GTGATTGTGCCGGTG GCGATTATTGCGGCG CCGACGCCCAAGAAC CAGGAAGCC 17 ALIVAIAPALVP ATTTATCATATGGCG CTGATTGTGGCGATT GCGCCGGCGCTGGTG CCGACGCCCAAGAAC CAGGAAGCC
TABLE-US-00012 TABLE 10 [PCR Primers for His-tagged BMP2 (LAP + MP) Proteins] Clone Primer sequence Name Abbreviation (5' → 3') 2L-1 HB2L Forward ATTTATCATATGCTCGTTCC GGAGCTGGGCCGC Reverse GGTATTGGATCCCTAGCGAC ACCCACA 2L-2 HM24B2L Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCTCGTTCCGGAG CTGGGCCGC Reverse GGTATTGGATCCCTAGCGAC ACCCACA 2L-3 HM24B2LSA Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCTCGTTCCGGAG CTGGGCCGC Reverse TATGTTGGATCCGTAGCGAC ACCCACA Forward CCCGGATCCATGGCAAATAT TACCGTTTTCTATAACGAA Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 2L-4 HM24B2LSB Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCTCGTTCCGGAG CTGGGCCGC Reverse TATGTTGGATCCGTAGCGAC ACCCACA Forward CCCGGATCCATGGCAGAACA AAGCGACAAGGATGTGAAG Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT 2L-5 HSBB2LSBM123 Forward TCTTGTCATATGGCAGAACA AAGCGACAAG Reverse TAAGTTGCGGCCGCTTACGC CAGCAGCGCCGCCGGCACAA TAATCGCCGCCGGAAGGGTT TCCGAAGG 2L-5C HSBB2LSB Forward TCTTGTCATATGGCAGAACA AAGCGACAAG Reverse AATAACGCGGCCGCTTAAAG GGTTTCCGAAGG 2L-6 HSAB2LSBM123 Forward GGGTTTCATATGATGGCAAA TATTACCGTTTTC Reverse TAAGTTGCGGCCGCTTACGC CAGCAGCGCCGCCGGCACAA TAATCGCCGCCGGAAGGGTT TCCGAAGG 2L-6C HSAB2LSB Forward GGGTTTCATATGATGGCAAA TATTACCGTTTTC Reverse AATAACGCGGCCGCTTAAAG GGTTTCCGAAGG 2L-7 SCHB2LM123 Forward AATATAGGATCCCTCGTTCC GGAGCTGGGC Reverse TATATTGTCGACTTACGCCA GCAGCGCCGCCGGCACAATA ATCGCCGCCGGGCGACACCC ACAACCCTC 2L-7C SCHB2L Forward AATATAGGATCCCTCGTTCC GGAGCTGGGC Reverse GTATTGGTCGACTTAGCGAC ACCCACAACC
TABLE-US-00013 TABLE 11 [PCR Primers for His-tagged BMP7 (LAP + MP) Proteins] Clone Primer sequence Name Abbreviation (5' → 3') 7L-1 HB7L Forward ATTTATCATATGTCCGCCCT GGCCGACTTCAGC Reverse ATAAATGGATCCCTAGTGGC AGCCACA 7L-2 HM24B7L Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCTCGTTCCGGAG CTGGGCCGC Reverse GGTATTGGATCCCTAGCGAC ACCCACA 7L-3 HM24B7LSA Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGTCCGCCCTGGCC GACTTCAGC Reverse ATAAATGGATCCGTAGTGGC AGCCACA Forward CCCGGATCCATGGCAAATAT TACCGTTTTCTATAACGAA Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 7L-4 HM24B7LSB Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGTCCGCCCTGGCC GACTTCAGC Reverse ATAAATGGATCCGTAGTGGC AGCCACA Forward CCCGGATCCATGGCAGAACA AAGCGACAAGGATGTGAAG Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT 7L-5 HSBB7LSBM123 Forward TCTTGTCATATGGCAGAACA AAGCGACAAG Reverse TAAGTTGCGGCCGCTTACGC CAGCAGCGCCGCCGGCACAA TAATCGCCGCCGGAAGGGTT TCCGAAGG 7L-5C HSBB7LSB Forward TCTTGTCATATGGCAGAACA AAGCGACAAG Reverse AATAACGCGGCCGCTTAAAG GGTTTCCGAAGG 7L-6 HM24SAB7LSBM123 Forward GGGTTTCATATGATGGCAAA TATTACCGTTTTC Reverse TAAGTTGCGGCCGCTTACGC CAGCAGCGCCGCCGGCACAA TAATCGCCGCCGGAAGGGTT TCCGAAGG 7L-6C HSAB7LSB Forward GGGTTTCATATGATGGCAAA TATTACCGTTTTC Reverse AATAACGCGGCCGCTTAAAG GGTTTCCGAAGG 7L-7 SCHB7LM123 Forward AATGATGGATCCTCCGCCCT GGCCGACTTC Reverse ATTTATGTCGACTTACGCCA GCAGCGCCGCCGGCACAATA ATCGCCGCCGGGTGGCAGCC ACAGGCCCG 7L-7C SCHB7L Forward AATGATGGATCCTCCGCCCT GGCCGACTTC Reverse TAATATGTCGACTTAGTGGC AGCCACAGGC
Example 2
Selection of Solubilization Domain for Recombinant Proteins
[0124] Recombinant cargo (BMP2 and BMP7) proteins fused to hydrophobic CPP could be expressed in the bacteria system, and purified with single-step affinity chromatography; however, protein is highly insoluble in physiological buffers (DMEM) and has extremely low yield as a soluble form. Therefore, an additional non-functional protein domain (solubilization domain: SD) has been applied to fuse with the recombinant protein to improve the solubility, yield and eventually cell and tissue permeability. According to the specific aim, the selected domains are SDA, SDB, SDC, SDD, SDE and SDF. The aMTD-/solubilization domain-fused recombinant protein is expected to be more stable and soluble.
[0125] Therefore, we hypothesize that SD and aMTDs do greatly influence in the improvement of solubility, yield and cell/tissue permeability of recombinant cargo proteins--BMP2/7--for further clinical application.
Example 3
Construction of Expression Vectors for Recombinant Proteins
[0126] Full-length cDNA for human BMP2 (RC214586) and BMP7 (RC203813) were purchased from Origene. New hydrophobic CPPs were identified by analyzing published hydrophobic CPP to optimize the critical factors for design of improved MTDs. For short form CP-BMPs (MP), aMTD24 was used, while the aMTD123 was used for the long form CP-BMPs (LAP+MP; LP). Coding sequences for aMTD-BMP2/7-SD fusion proteins were cloned into pET28a(+) from PCR-amplified DNA segments. BMP2- and 7-fused recombinant proteins were expressed in E. coli BL21-CodonPlus (DE3) from pET28a (+)-based plasmid. These E. coli cells were grown to an A600 of 0.4˜0.5 and induced for 3 hours with 0.7 mM IPTG. The 6×histidine-tagged recombinant BMP2 and BMP7 proteins were purified by Ni2+-affinity chromatography under the denaturing conditions and refolded by dialyzing with refolding buffer. After the purification, proteins were dialyzed with physiological buffer. PCR primers for the His-tagged BMP2 and BMP7 recombinant proteins fused to aMTD and SD are summarized in TABLES 5, 6, 9, and 10.
TABLE-US-00014 TABLE 6 [PCR Primers for His-tagged BMP2 (MP) Proteins] Clone Primer sequence Name Abbreviation (5' → 3') 2M-1 HB2M Forward ATTTATCATATGCAAGCCAA ACACAAACAGCGG Reverse GGTATTGGATCCCTAGCGAC ACCCACA 2M-2 HM24B2M Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCAAGCCAAACAC AAACAGCGG Reverse GGTATTGGATCCCTAGCGAC ACCCACA 2M-3 HM24B2MSA Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCAAGCCAAACAC AAACAGCGG Reverse TATGTTGGATCCGTAGCGAC ACCCACA Forward CCCGGATCCATGGCAAATAT TACCGTTTTCTATAACGAA Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 2M-3C HB2MSA Forward ATTTATCATATGCAAGCCAA ACACAAACAGCGG Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 2M-4 HM24B2MSB Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGCAAGCCAAACAC AAACAGCGG Reverse TATGTTGGATCCGTAGCGAC ACCCACA Forward CCCGGATCCATGGCAGAACA AAGCGACAAGGATGTGAAG Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT 2M-4C HB2MSB Forward ATTTATCATATGCAAGCCAA ACACAAACAGCGG Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT
Example 4
Construction of Expression Vectors for Cell-/Tissue-Permeability Optimization of aMTD-Fused BMP2/7 Recombinant Proteins
[0127] Eleven kinds of a MTD sequences were selected from 240 aMTD pool (TABLE 2) which were designed based on 6 critical factors. Construction of expression vectors were performed as described in Example 3. PCR primers for the His-tagged BMP2 and BMP7 recombinant proteins fused to 17 kinds of aMTDs are summarized in TABLES 7 and 8.
TABLE-US-00015 TABLE 7 [PCR Primers for His-tagged BMP7 (MP) Proteins] Clone Primer sequence Name Abbreviation (5' → 3') 7M-1 HB7M Forward ATTTATCATATGACGCCCAA GAACCAGGAAGCC Reverse ATAAATGGATCCCTAGTGGC AGCCACA 7M-2 HM24B7M Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGACGCCCAAGAAC CAGGAAGCC Reverse ATAAATGGATCCGTAGTGGC AGCCACA 7M-3 HM24B7MSA Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGACGCCCAAGAAC CAGGAAGCC Reverse ATAAATGGATCCGTAGTGGC AGCCACA Forward CCCGGATCCATGGCAAATAT TACCGTTTTCTATAACGAA Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 7M-3C HB7MSA Forward ATTTATCATATGACGCCCAA GAACCAGGAAGCC Reverse CGCGTCGACTTACCTCGGCT GCACCGGCACGGAGATGAC 7M-4 HM24B7MSB Forward ATTTATCATATGATTGCGCT GGCGGCGCCGGCGCTGATTG TGGCGCCGACGCCCAAGAAC CAGGAAGCC Reverse ATAAATGGATCCGTAGTGGC AGCCACA Forward CCCGGATCCATGGCAGAACA AAGCGACAAGGATGTGAAG Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT 7M-4C HB7MSB Forward ATTTATCATATGACGCCCAA GAACCAGGAAGCC Reverse CGCGTCGACTTAAAGGGTTT CCGAAGGCTTGGCTATCTT
TABLE-US-00016 TABLE 8 [PCR Primers for His-tagged BMP2 (MP) Proteins Fused to Different 17 Kinds of aMTDs] A/a 5' Primer 3' Primer ID Sequence (5' → 3') (5' → 3') 1 AAALAPVVLALP ATTTATCATATGGCG CGCGTCGAC GCGGCGCTGGCGCCG TTACCTCGG GTGGTGCTGGCGCTG CTGCACCGG CCGCAAGCCAAACAC CACGGAGAT AAACAGCGG GAC 2 AALLVPAAVLAP ATTTATCATATGGCG GCGCTGCTGGTGCCG GCGGCGGTGCTGGCG CCGCAAGCCAAACAC AAACAGCGG 3 VAALPVLLAALP ATTTATCATATGGTG GCGGCGCTGCCGGTG CTGCTGGCGGCGCTG CCGCAAGCCAAACAC AAACAGCGG 4 AAAVVPVLLVAP ATTTATCATATGGCG GCGGCGGTGGTGCCG GTGCTGCTGGTGGCG CCGCAAGCCAAACAC AAACAGCGG 5 IVAIAVPALVAP ATTTATCATATGATT GTGGCGATTGCGGTG CCGGCGCTGGTGGCG CCGCAAGCCAAACAC AAACAGCGG 6 AAALVIPAAILP ATTTATCATATGGCG GCGGCGCTGGTGATT CCGGCGGCGATTCTG CCGCAAGCCAAACAC AAACAGCGG 7 ALAALVPAVLVP ATTTATCATATGGCG CTGGCGGCGCTGGTG CCGGCGGTGCTGGTG CCGCAAGCCAAACAC AAACAGCGG 8 VLVALAAPVIAP ATTTATCATATGGTG CTGGTGGCGCTGGCG GCGCCGGTGATTGCG CCGCAAGCCAAACAC AAACAGCGG 9 IVAVALPALAVP ATTTATCATATGATT GTGGCGGTGGCGCTG CCGGCGCTGGCGGTG CCGCAAGCCAAACAC AAACAGCGG 10 IAVALPALIAAP ATTTATCATATGATT GCGGTGGCGCTGCCG GCGCTGATTGCGGCG CCGCAAGCCAAACAC AAACAGCGG 11 ALAVIVVPALAP ATTTATCATATGGCG CTGGCGGTGATTGTG GTGCCGGCGCTGGCG CCGCAAGCCAAACAC AAACAGCGG 12 AVVIALPAVVAP ATTTATCATATGGCG GTGGTGATTGCGCTG CCGGCGGTGGTGGCG CCGCAAGCCAAACAC AAACAGCGG 13 LVAIVVLPAVAP ATTTATCATATGCTG GTGGCGATTGTGGTG CTGCCGGCGGTGGCG CCGCAAGCCAAACAC AAACAGCGG 14 AIAIAIVPVALP ATTTATCATATGGCG ATTGCGATTGCGATT GTGCCGGTGGCGCTG CCGCAAGCCAAACAC AAACAGCGG 15 VAAAIALPAIVP ATTTATCATATGGTG GCGGCGGCGATTGCG CTGCCGGCGATTGTG CCGCAAGCCAAACAC AAACAGCGG 16 AVIVPVAIIAAP ATTTATCATATGGCG GTGATTGTGCCGGTG GCGATTATTGCGGCG CCGCAAGCCAAACAC AAACAGCGG 17 ALIVAIAPALVP ATTTATCATATGGCG CTGATTGTGGCGATT GCGCCGGCGCTGGTG CCGCAAGCCAAACAC AAACAGCGG
Example 5
Protein Labeling and Analysis of Protein Uptake in Cultured Cells
[0128] Recombinant proteins were conjugated to fluorescein isothiocynate (FITC), according to the manufacturer's instructions (Sigma, F7250). RAW 264.7 were treated with 10 μM FITC-labeled proteins (FITC-2M-1, FITC-2M-2, FITC-2M-3, FITC-2M-4, FITC-7M-1, FITC-7M-2, FITC-7M-3 and FITC-7M-4) or unconjugated FITC (FITC only) for 1 hour at 37° C., washed 2 times with PBS, treated with proteinase K (10 μg/mL) for 20 minutes at 37° C. to remove cell-surface bound proteins and subjected to FACS analysis (Guava easyCyte 8, Millipore). To visualize protein uptake, they were conducted in much the same manner, except NIH3T3 cells, where they were exposed to 10 μM FITC-proteins for 1 hour at 37° C., and their nuclei were stained for DAPI. Cells were washed 3 times with PBS after exposing them in the mounting solution and examined by confocal laser scanning microscopy (Zeiss, LSM 700).
Example 6
Tissue Distribution of CP-BMP2/7 Proteins
[0129] ICR mice (6-week-old, male) were injected intraperitoneally (600 μg/head) with FITC only or FITC-conjugated proteins (FITC-2M-4C, FITC-2M-4, FITC-7M-4 and FITC-7M4C). After 2 hours, the organs (brain, heart, lung, liver, spleen and kidney) were isolated, washed with O.C.T. compound (Sakura), and frozen in deep freezer. Cryosections (15 μm thickness) were analyzed by fluorescence microscopy.
Example 7
Cell Culture and Osteogenic Differentiation
[0130] 7-1. Cell Culture C2C12 cells were cultured with high glucose DMEM (Hyclone) and 10% fetal bovine serum (FBS) at 37° C. for growth and expansion. For ALP assay and morphology observation, C2C12 myoblasts were plated on 24-well culture plate (1×105 cells/well) in the growth media for 24 hours. Mouse pre-osteoblast, MC3T3-E1 cells were cultured in the minimum essential medium (MEM). Alpha Modification and C3H10T1/2 mesenchymal stem cells were maintained in the Roswell Park Memorial Institute medium (RPMI) 1640 with 10% FBS and 1% penicillin/streptomycin.
[0131] 7-2. Differentiation of Cells
[0132] To induce the differentiation, cells were exposed to a starvation condition with 2% of FBS in a culture media with or without CP-BMPs. Proteins were treated with different concentration and treatment to follow the purpose of each experiment. After 3 days and 7 days of culture, cell morphologies were photographed to determine the differentiation into either myotube formation or osteogenesis.
[0133] 7-3. Phosphorylation of Smad Signaling
[0134] Preosteoblasts (MC3T3E1), myoblasts (C2C12), and multiple mesenchymal stem cell (C3H/10T1/2) are incubated with serum-free medium alone (αMEM or DMEM) containing 10 μM CP-BMP2 and CP-BMP7 proteins of indicated concentration during various time. To investigate the activation of BMP-Smad signaling, treated CP-BMP2 and CP-BMP7 cells were lysed in a lysis buffer (RIPA buffer) containing a protease cocktail and phosphatase inhibitor cocktail. Equal amounts of cell lysate protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The protein transferred membranes were incubated to block non-specific binding sites in immersing the membrane in 5% non-fat dried milk. The membranes were incubated with anti-phosphorylated Smad1/5/8 overnight at 4° C. and anti-β-actin at room temperature (RT) and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour at RT. The blots were developed using a chemiluminescence detection system and exposed to an x-ray film.
[0135] 7-4. Measurement of Alkaline Phosphatase Activity
[0136] ALP activity was measured with cell lysate, according to the manufacturer's protocol. Briefly, supernatant of cell lysate was used after 13000 rpm of centrifugation for 10 min, and 10 μl of supernatant was reacted with 200 μl of ALP substrate solution for 30 minutes at 37° C. After 30 minutes, the optical density (O.D) was measured by using microplate reader at 405 nm of wave length. Various concentrations of p-Nitrophenyl Phosphate were used as standards for ALP activity, and calculated ALP activities were normalized by total protein concentration, which was obtained from bradford (Bio-rad) protein assay.
[0137] 7-5. Measurement of Calcium Content
[0138] To determine the calcium deposition in extra cellular matrix (ECM) after treatment with CP-BMPs, the cells were washed with PBS 3 times then added 300 μl of 0.6 N HCl and incubated in deep freezer for 24 hours to extract calcium. Calcium content was quantified using QuantiChrom Calcium Assay kits (Bioassay Systems, Hayward, Calif., USA) as manufacturer's instruction. Briefly, 5 μl of each sample was placed in 96-well plate and reacted with 200 pl of working reagent. After 3 minutes, optical density was measured at 612 nm wave length.
[0139] 7-6. Alizarin Red S Staining
[0140] MC3T3-E1 cells and C3H10T1/2 cells were plated at 5×104 cells per well in 24-well plate and cultured with a-MEM containing 10% FBS and 1% penicillin/streptomycin. Confluent MC3T3-E1 cells were treated with ascorbic acid (Sigma-Aldrich; 50 mg/mL) and 5 mM β-glycophosphate including CP-BMP2 and CP-BMP7. To induce osteogenic differentiation in confluent C3H10T1/2 cells, osteogenic medium including 0.1 μM dexamethasone and 10 mM β-glycophosphate were treated with or without CP-BMP2 and CP-BMP7. After 21 days, mineralization of bone nodules was detected in cultured cells by alizarin red staining. The cells were washed with PBS, and fixed with 4% paraformaldehyde and then stained with 0.4M alizarin red S, pH 4.2, for 10 minutes at RT.
Example 8
Preclinical Models (In Vivo)
[0141] 8-1. In Vivo Calvarial Critical Sized Defect Model
[0142] The effect of CP-BMP2/7 on in vivo bone regeneration was investigated by calvarial critical sized defect model using 6-week-old ICR mice (Dooyeol biothec, Seoul, Korea). Mice were anesthetized with Zoletil (60 mg/kg) and Xylazine (20 mg/kg) and exposed incision area by shaving scalp hair. For defect creation, head skin incision was performed; two defects on both sides of the calvaria were made by using 4 mm-diameter surgical trephine bur. Surgery sites were sutured and treated with Povidone iodine. After 24 hours of surgery, the recombinant CP-BMPs were locally injected to surgery site, and the injection was repeated by weekly during experimental periods. All mice were sacrificed after 8 weeks and calvaria tissues were fixed with 10% formalin solution at 4° C. for 3 days for further examinations.
[0143] 8-2. Calvarial Injection Assay
[0144] To confirm the effect of new bone formation of CP-BMPs or vehicle, recombinant proteins were daily treated to calvarial bones of mice by subcutaneous simple injection for 4 weeks. After 4 weeks, we dissected out the calvarial bones and fixed tissues within 4% paraformaldehyde. Decalcified calvarial bones were embedded with paraffin and cut 3-μm sections on a microtome. To confirm new formation of calcified bone, sections were stained Goldner's trichrome as described in `4.5.5 Histological analysis` section.
[0145] 8-3. Soft X-Ray
[0146] To determine the bone regeneration in calvarial critical sized defect model, the fixed calvarial tissues were exposed to soft X-rays (CMP-2, Softex Co., Tokyo, Japan) under optimized exposure condition (23 kV, 2 mA, 90 s). The exposed results were obtained by the developing film.
[0147] 8-4. 3D micro-CT
[0148] Three-dimensional images from micro-CT scanning were analyzed with Adobe Photoshop CS6 (Adobe Systems, CA, USA) to measure regenerated bone areas.
[0149] 8-5. Histological Analysis Samples were decalcified using Rapidcal for 2 weeks (BBC Biochemical, Mount Vernon, Wash., USA) by replasing the solution every 2 days. Samples were dehydrated with graded EtOH (70-100%), toluene, and paraffin. Dehydrated samples were embedded in paraffin wax and hardened into a paraffin block for sectioning. Specimens were cut to 6 μm using a microtome (Shandon, Runcorn, Cheshire, GB). Sections underwent deparaffinization and hydration and stained nuclei and cytosol with Harris hematoxylin and eosin solution. Goldner's trichrome staining method was used to determined detailed bone tissue morphology such as mineralized collagen. Following dehydration, samples were mounted with mounting medium (Richard-Allan Scientific, Kalamazoo, Mich., USA) and observed under an optical microscope (Nikon 2000, Japan).
[0150] It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided that they come within the scope of the appended claims and their equivalents.
TABLE-US-00017 [cDNA Sequence of Histidine Tag] SEQ ID NO: 481 ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGG TGCCGCGCGGCAGC [Amino Acid Sequence of Histidine Tag] SEQ ID NO: 482 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser [cDNA Sequence of human BMP2] SEQ ID NO: 483 ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCC AGGTCCTCCTGGGCGGCGCGGCTGGCCTCGTTCCGGAGCTGGG CCGCAGGAAGTTCGCGGCGGCGTCGTCGGGCCGCCCCTCATCC CAGCCCTCTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGC TCAGCATGTTCGGCCTGAAACAGAGACCCACCCCCAGCAGGGA CGCCGTGGTGCCCCCCTACATGCTAGACCTGTATCGCAGGCAC TCGGGTCAGCCGGGCTCACCCGCCCCAGACCACCGGTTGGAGA GGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACCATGA AGAATCTTTGGAAGAACTACCAGAAACGAGTGGGAAAACAACC CGGAGATTCTTCTTTAATTTAAGTTCTATCCCCACGGAGGAGT TTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCA AGATGCTTTAGGAAACAATAGCAGTTTCCATCACCGAATTAAT ATTTATGAAATCATAAAACCTGCAACAGCCAACTCGAAATTCC CCGTGACCAGTCTTTTGGACACCAGGTTGGTGAATCAGAATGC AAGCAGGTGGGAAAGTTTTGATGTCACCCCCGCTGTGATGCGG TGGACTGCACAGGGACACGCCAACCATGGATTCGTGGTGGAAG TGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACATGT TAGGATAAGCAGGTCTTTGCACCAAGATGAACACAGCTGGTCA CAGATAAGGCCATTGCTAGTAACTTTTGGCCATGATGGAAAAG GGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCCAAACACAA ACAGCGGAAACGCCTTAAGTCCAGCTGTAAGAGACACCCTTTG TACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATTGTGG CTCCCCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGCCC TTTTCCTCTGGCTGATCATCTGAACTCCACTAATCATGCCATT GTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGG CATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTGTA CCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGAC ATGGTTGTGGGCTAG [Amino Acid Sequence of human BMP2] SEQ ID NO: 484 Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg [cDNA Sequence of Human BMP7] SEQ ID NO: 485 ATGCACGTGCGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCG TGGCGCTCTGGGCACCCCTGTTCCTGCTGCGCTCCGCCCTGGC CGACTTCAGCCTGGACAACGAGGTGCACTCGAGCTTCATCCAC CGGCGCCTCCGCAGCCAGGAGCGGCGGGAGATGCAGCGCGAGA TCCTCTCCATTTTGGGCTTGCCCCACCGCCCGCGCCCGCACCT CCAGGGCAAGCACAACTCGGCACCCATGTTCATGCTGGACCTG TACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGCGGCC AGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGG CCCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGAC GCCGACATGGTCATGAGCTTCGTCAACCTCGTGGAACATGACA AGGAATTCTTCCACCCACGCTACCACCATCGAGAGTTCCGGTT TGATCTTTCCAAGATCCCAGAAGGGGAAGCTGTCACGGCAGCC GAATTCCGGATCTACAAGGACTACATCCGGGAACGCTTCGACA ATGAGACGTTCCGGATCAGCGTTTATCAGGTGCTCCAGGAGCA CTTGGGCAGGGAATCGGATCTCTTCCTGCTCGACAGCCGTACC CTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGACATCACAG CCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTGGG CCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAAC CCCAAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAACA AGCAGCCCTTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCA CTTCCGCAGCATCCGGTCCACGGGGAGCAAACAGCGCAGCCAG AACCGCTCCAAGACGCCCAAGAACCAGGAAGCCCTGCGGATGG CCAACGTGGCAGAGAACAGCAGCAGCGACCAGAGGCAGGCCTG TAAGAAGCACGAGCTGTATGTCAGCTTCCGAGACCTGGGCTGG CAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACT GTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGC CACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAAC CCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCA ATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCAT CCTGAAGAAATACAGAAACATGGTGGTCCGGGCCTGTGGCTGC CACTAG [Amino Acid Sequence of Human BMP7] SEQ ID NO: 486 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu
Glu Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His [cDNA Sequences of SDA] SEQ ID NO: 487 ATGGCAAATA TTACCGTTTT CTATAACGAA GACTTCCAGG GTAAGCAGGT CGATCTGCCG CCTGGCAACT ATACCCGCGC CCAGTTGGCG GCGCTGGGCA TCGAGAATAA TACCATCAGC TCGGTGAAGG TGCCGCCTGG CGTGAAGGCT ATCCTGTACC AGAACGATGG TTTCGCCGGC GACCAGATCG AAGTGGTGGC CAATGCCGAG GAGTTGGGCC CGCTGAATAA TAACGTCTCC AGCATCCGCG TCATCTCCGT GCCCGTGCAG CCGCGCATGG CAAATATTAC CGTTTTCTAT AACGAAGACT TCCAGGGTAA GCAGGTCGAT CTGCCGCCTG GCAACTATAC CCGCGCCCAG TTGGCGGCGC TGGGCATCGA GAATAATACC ATCAGCTCGG TGAAGGTGCC GCCTGGCGTG AAGGCTATCC TCTACCAGAA CGATGGTTTC GCCGGCGACC AGATCGAAGT GGTGGCCAAT GCCGAGGAGC TGGGTCCGCT GAATAATAAC GTCTCCAGCA TCCGCGTCAT CTCCGTGCCG GTGCAGCCGA GG [Amino Acid Sequences of SDA] SEQ ID NO: 488 Met Ala Asn Ile Thr Val Phe Tyr Asn Glu Asp Phe Gln Gly Lys Gln Val Asp Leu Pro Pro Gly Asn Tyr Thr Arg Ala Gln Leu Ala Ala Leu Gly Ile Glu Asn Asn Thr Ile Ser Ser Val Lys Val Pro Pro Gly Val Lys Ala Ile Leu Tyr Gln Asn Asp Gly Phe Ala Gly Asp Gln Ile Glu Val Val Ala Asn Ala Glu Glu Leu Gly Pro Leu Asn Asn Asn Val Ser Ser Ile Arg Val Ile Ser Val Pro Val Gln Pro Arg Met Ala Asn Ile Thr Val Phe Tyr Asn Glu Asp Phe Gln Gly Lys Gln Val Asp Leu Pro Pro Gly Asn Tyr Thr Arg Ala Gln Leu Ala Ala Leu Gly ile Glu Asn Asn Thr Ile Ser Ser Val Lys Val Pro Pro Gly Val Lys Ala Ile Leu Tyr Gln Asn Asp Gly Phe Ala Gly Asp Gln Ile Glu Val Val Ala Asn Ala Glu Glu Leu Gly Pro Leu Asn Asn Asn Val Ser Ser Ile Arg Val Ile Ser Val Pro Val Gln Pro Arg [cDNA Sequences of SDB] SEQ ID NO: 489 ATGGCAGAAC AAAGCGACAA GGATGTGAAG TACTACACTC TGGAGGAGAT TCAGAAGCAC AAAGACAGCA AGAGCACCTG GGTGATCCTA CATCATAAGG TGTACGATCT GACCAAGTTT CTCGAAGAGC ATCCTGGTGG GGAAGAAGTC CTGGGCGAGC AAGCTGGGGG TGATGCTACT GAGAACTTTG AGGACGTCGG GCACTCTACG GATGCACGAG AACTGTCCAA AACATACATC ATCGGGGAGC TCCATCCAGA TGACAGATCA AAGATAGCCA AGCCTTCGGA AACCCTT [Amino Acid Sequences of SDB] SEQ ID NO: 450 Met Ala Glu Gln Ser Asp Lys Asp Val Lys Tyr Tyr Thr Leu Glu Glu Ile Gln Lys His Lys Asp Ser Lys Ser Thr Trp Val Ile Leu His His Lys Val Tyr Asp Leu Thr Lys Phe Leu Glu Glu His Pro Gly Gly Glu Glu Val Leu Gly Glu Gln Ala Gly Gly Asp Ala Thr Glu Asn Phe Glu Asp Val Gly His Ser Thr Asp Ala Arg Glu Leu Ser Lys Thr Tyr Ile Ile Gly Glu Leu His Pro Asp Asp Arg Ser Lys Ile Ala Lys Pro Ser Glu Thr Leu [cDNA Sequences of SDC] SEQ ID NO: 451 ATGAGCGATA AAATTATTCA CCTGACTGAC GACAGTTTTG ACACGGATGT ACTCAAAGCG GACGGGGCGA TCCTCGTCGA TTTCTGGGCA GAGTGGTGCG GTCCGTGCAA AATGATCGCC CCGATTCTGG ATGAAATCGC TGACGAATAT CAGGGCAAAC TGACCGTTGC AAAACTGAAC ATCGATCAAA ACCCTGGCAC TGCGCCGAAA TATGGCATCC GTGGTATCCC GACTCTGCTG CTGTTCAAAA ACGGTGAAGT GGCGGCAACC AAAGTGGGTG CACTGTCTAA AGGTCAGTTG AAAGAGTTCC TCGACGCTAA CCTGGCC [Amino Acid Sequences of SDC] SEQ ID NO: 452 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala [cDNA Sequences of SDD] SEQ ID NO: 453 ATGAAAAAGA TTTGGCTGGC GCTGGCTGGT TTAGTTTTAG CGTTTAGCGC ATCGGCGGCG CAGTATGAAG ATGGTAAACA GTACACTACC CTGGAAAAAC CGGTAGCTGG CGCGCCGCAA GTGCTGGAGT TTTTCTCTTT CTTCTGCCCG CACTGCTATC AGTTTGAAGA AGTTCTGCAT ATTTCTGATA ATGTGAAGAA AAAACTGCCG GAAGGCGTGA AGATGACTAA ATACCACGTC AACTTCATGG GTGGTGACCT GGGCAAAGAT CTGACTCAGG CATGGGCTGT GGCGATGGCG CTGGGCGTGG AAGACAAAGT GACTGTTCCG CTGTTTGAAG GCGTACAGAA AACCCAGACC ATTCGTTCTG CTTCTGATAT CCGCGATGTA TTTATCAACG CAGGTATTAA AGGTGAAGAG TACGACGCGG CGTGGAACAG CTTCGTGGTG AAATCTCTGG TCGCTCAGCA GGAAAAAGCT GCAGCTGACG TGCAATTGCG TGGCGTTCCG GCGATGTTTG TTAACGGTAA ATATCAGCTG AATCCGCAGG GTATGGATAC CAGCAATATG GATGTTTTTG TTCAGCAGTA TGCTGATACA GTGAAATATC TGTCCGAGAA AAAA [Amino Acid Sequences of SDD] SEQ ID NO: 454 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser Ala Ser Ala Ala Gln Tyr Glu Asp Gly Lys Gln Tyr Thr Thr Leu Glu Lys Pro Val Ala Gly Ala Pro Gln Val Leu Glu Phe Phe Ser Phe Phe Cys Pro His Cys Tyr Gln Phe Glu Glu Val Leu His Ile Ser Asp Asn Val Lys Lys Lys Leu Pro Glu Gly Val Lys Met Thr Lys Tyr His Val Asn Phe Met Gly Gly Asp Leu Gly Lys Asp Leu Thr Gln Ala Trp Ala Val Ala Met Ala Leu Gly Val Glu Asp Lys Val Thr Val Pro Leu Phe Glu Gly Val Gln Lys Thr Gln Thr Ile Arg Ser Ala Ser Asp Ile Arg Asp Val Phe Ile Asn Ala Gly Ile Lys Gly Glu Glu Tyr Asp Ala Ala Trp Asn Ser Phe Val Val Lys Ser Leu Val Ala Gln Gln Glu Lys Ala Ala Ala Asp Val Gln
Leu Arg Gly Val Pro Ala Met Phe Val Asn Gly Lys Tyr Gln Leu Asn Pro Gln Gly Met Asp Thr Ser Asn Met Asp Val Phe Val Gln Gln Tyr Ala Asp Thr Val Lys Tyr Leu Ser Glu Lys Lys [cDNA Sequences of SDE] SEQ ID NO: 456 GGGTCCCTGC AGGACTCAGA AGTCAATCAA GAAGCTAAGC CAGAGGTCAA GCCAGAAGTC AAGCCTGAGA CTCACATCAA TTTAAAGGTG TCCGATGGAT CTTCAGAGAT CTTCTTCAAG ATCAAAAAGA CCACTCCTTT AAGAAGGCTG ATGGAAGCGT TCGCTAAAAG ACAGGGTAAG GAAATGGACT CCTTAACGTT CTTGTACGAC GGTATTGAAA TTCAAGCTGA TCAGACCCCT GAAGATTTGG ACATGGAGGA TAACGATATT ATTGAGGCTC ACCGCGAACA GATTGGAGGT [Amino Acid Sequences of SDE] SEQ ID NO: 457 Gly Ser Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Thr Phe Leu Tyr Asp Gly Ile Glu Ile Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly [cDNA Sequences of SDF] SEQ ID NO: 458 GGATCCGAAA TCGGTACTGG CTTTCCATTC GACCCCCATT ATGTGGAAGT CCTGGGCGAG CGCATGCACT ACGTCGATGT TGGTCCGCGC GATGGCACCC CTGTGCTGTT CCTGCACGG AACCCGACCT CCTCCTACGT GTGGCGCAAC ATCATCCCGC ATGTTGCACC GACCCATCGC TGCATTGCTC CAGACCTGAT CGGTATGGGC AAATCCGACA AACCAGACCT GGGTTATTTC TTCGACGACC ACGTCCGCTT CATGGATGCC TTCATCGAAG CCCTGGGTCT GGAAGAGGTC GTCCTGGTCA TTCACGACTG GGGCTCCGCT CTGGGTTTCC ACTGGGCCAA GCGCAATCCA GAGCGCGTCA AAGGTATTGC ATTTATGGAG TTCATCCGCC CTATCCCGAC CTGGGACGAA TGGCCAGAAT TTGCCCGCGA GACCTTCCAG GCCTTCCGCA CCACCGACGT CGGCCGCAAG CTGATCATCG ATCAGAACGT TTTTATCGAG GGTACGCTGC CGATGGGTGT CGTCCGCCCG CTGACTGAAG TCGAGATGGA CCATTACCGC GAGCCGTTCC TGAATCCTGT TGACCGCGAG CCACTGTGGC GCTTCCCAAA CGAGCTGCCA ATCGCCGGTG AGCCAGCGAA CATCGTCGCG CTGGTCGAAG AATACATGGA CTGGCTGCAC CAGTCCCCTG TCCCGAAGCT GCTGTTCTGG GGCACCCCAG GCGTTCTGAT CCCACCGGCC GAAGCCGCTC GCCTGGCCAA AAGCCTGCCT AACTGCAAGG CTGTGGACAT CGGCCCGGGT CTGAATCTGC TGCAAGAAGA CAACCCGGAC CTGATCGGCA GCGAGATCGC GCGCTGGCTG TCTACTCTGG AGATTTCCGGT [Amino Acid Sequences of SDF] SEQ ID NO: 459 Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly
REFERENCES
[0151] 1. Soltanoff C S, Yang S, Chen W, Li Y R Signaling networks that control the lineage commitment and differentiation of bone cells. Crit Rev Eukaryot Gene Expr 2009; 19(1):1-46.
[0152] 2. Kawabata M, Imamura T, Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev 1998; 9(1):49-61.
[0153] 3. Carreira A C, Alves G G, Zambuzzi W F, Sogayar M C, Granjeiro J M. Bone Morphogenetic Proteins: structure, biological function and therapeutic applications.
[0154] Arch Biochem Biophys 2014; 561:64-73.
[0155] 4. ten Dijke P, Fu J, Schaap P, Roelen B A. Signal transduction of bone morphogenetic proteins in osteoblast differentiation. J Bone Joint Surg Am 2003; 85-A Suppl 3:34-8.
[0156] 5. Canalis E, Economides A N, Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev 2003; 24(2):218-35.
[0157] 6. Huang Z, Ren P G, Ma T, Smith R L, Goodman S B. Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability. Cytokine 2010; 51(3):305-10.
[0158] 7. Noel D, Gazit D, Bouquet C, Apparailly F, Bony C, Plence P, et al. Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells. Stem Cells 2004; 22(1):74-85.
[0159] 8. Shen B, Wei A, Whittaker S, Williams L A, Tao H, Ma D D, et al. The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro. J Cell Biochem 2010; 109(2):406-16.
[0160] 9. Weiskirchen R, Meurer S K. BMP-7 counteracting TGF-beta1 activities in organ fibrosis. Front Biosci (Landmark Ed) 2013; 18:1407-34.
[0161] 10. Kudo T A, Kanetaka H, Watanabe A, Okumoto A, Asano M, Zhang Y, et al. Investigating bone morphogenetic protein (BMP) signaling in a newly established human cell line expressing BMP receptor type II. Tohoku J Exp Med 2010; 222(2):121-9.
[0162] 11. Liu H, Zhang R, Chen D, Oyajobi B O, Zhao M. Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation. J Cell Physiol 2012; 227(3):952-63.
[0163] 12. Zhang X, Schwarz E M, Young D A, Puzas J E, Rosier R N, O'Keefe R J. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002; 109(11):1405-15.
[0164] 13. van der Kraan P M, de Vries B J, Vitters E L, van den Berg W B, van de Putte L B. The effect of low sulfate concentrations on the glycosaminoglycan synthesis in anatomically intact articular cartilage of the mouse. J Orthop Res 1989; 7(5):645-53.
[0165] 14. Hunziker E B, Schenk R K, Cruz-Orive L M. Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg Am 1987; 69(2):162-73.
[0166] 15. Urist M R. Bone: formation by autoinduction. Science 1965; 150(3698):893-9.
[0167] 16. Khattab H M, Ono M, Sonoyama W, Oida Y, Shinkawa S, Yoshioka Y, et al. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism. Bone 2014; 69:165-73.
[0168] 17. Shim J H, Greenblatt M B, Singh A, Brady N, Hu D, Drapp R, et al. Administration of BMP2/7 in utero partially reverses Rubinstein-Taybi syndrome-like skeletal defects induced by Pdk1 or Cbp mutations in mice. J Clin Invest 2012; 122(1):91-106.
[0169] 18. Yasko A W, Lane J M, Fellinger E J, Rosen V, Wozney J M, Wang E A. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am 1992; 74(5):659-70.
[0170] 19. Einhorn T A, Majeska R J, Mohaideen A, Kagel E M, Bouxsein M L, Turek T J, et al. A single percutaneous injection of recombinant human bone morphogenetic protein-2 accelerates fracture repair. J Bone Joint Surg Am 2003; 85-A(8):1425-35.
[0171] 20. Nakase T, Nomura S, Yoshikawa H, Hashimoto J, Hirota S, Kitamura Y, et al. Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J Bone Miner Res 1994; 9(5):651-9.
[0172] 21. Balint E, Lapointe D, Drissi H, van der Meijden C, Young D W, van Wijnen A J, et al. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation. J Cell Biochem 2003; 89(2):401-26.
[0173] 22. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng J M, Behringer R R, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002; 108(1):17-29.
[0174] 23. Wegman F, Bijenhof A, Schuijff L, Oner F C, Dhert W J, Alblas J. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. European cells & materials 2011; 21:230-42; discussion 42.
Sequence CWU
1
1
480112PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 1Ala Ala Ala Leu
Ala Pro Val Val Leu Ala Leu Pro 1 5 10
212PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 2Ala Ala Ala Val
Pro Leu Leu Ala Val Val Val Pro 1 5 10
312PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 3Ala Ala Leu Leu
Val Pro Ala Ala Val Leu Ala Pro 1 5 10
412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 4Ala Leu Ala Leu
Leu Pro Val Ala Ala Leu Ala Pro 1 5 10
512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 5Ala Ala Ala Leu
Leu Pro Val Ala Leu Val Ala Pro 1 5 10
612PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 6Val Val Ala Leu
Ala Pro Ala Leu Ala Ala Leu Pro 1 5 10
712PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 7Leu Leu Ala Ala
Val Pro Ala Val Leu Leu Ala Pro 1 5 10
812PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 8Ala Ala Ala Leu
Val Pro Val Val Ala Leu Leu Pro 1 5 10
912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 9Ala Val Ala Leu
Leu Pro Ala Leu Leu Ala Val Pro 1 5 10
1012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 10Ala Val Val
Leu Val Pro Val Leu Ala Ala Ala Pro 1 5 10
1112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 11Val
Val Leu Val Leu Pro Ala Ala Ala Ala Val Pro 1 5
10 1212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 12Ile Ala Leu Ala Ala Pro Ala Leu Ile Val Ala Pro 1
5 10 1312PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 13Ile Val Ala Val Ala Pro Ala Leu Val Ala Leu Pro 1
5 10 1412PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 14Val Ala Ala Leu Pro Val Val Ala
Val Val Ala Pro 1 5 10
1512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 15Leu Leu Ala Ala
Pro Leu Val Val Ala Ala Val Pro 1 5 10
1612PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 16Ala Leu Ala
Val Pro Val Ala Leu Leu Val Ala Pro 1 5 10
1712PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 17Val
Ala Ala Leu Pro Val Leu Leu Ala Ala Leu Pro 1 5
10 1812PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 18Val Ala Leu Leu Ala Pro Val Ala Leu Ala Val Pro 1
5 10 1912PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 19Ala Ala Leu Leu Val Pro Ala Leu Val Ala Val Pro 1
5 10 2012PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 20Ala Ile Val Ala Leu Pro Val Ala
Val Leu Ala Pro 1 5 10
2112PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 21Ile Ala Ile Val
Ala Pro Val Val Ala Leu Ala Pro 1 5 10
2212PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 22Ala Ala Leu
Leu Pro Ala Leu Ala Ala Leu Leu Pro 1 5 10
2312PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 23Ala
Val Val Leu Ala Pro Val Ala Ala Val Leu Pro 1 5
10 2412PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 24Leu Ala Val Ala Ala Pro Leu Ala Leu Ala Leu Pro 1
5 10 2512PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 25Ala Ala Val Ala Ala Pro Leu Leu Leu Ala Leu Pro 1
5 10 2612PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 26Leu Leu Val Leu Pro Ala Ala Ala
Leu Ala Ala Pro 1 5 10
2712PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 27Leu Val Ala Leu
Ala Pro Val Ala Ala Val Leu Pro 1 5 10
2812PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 28Leu Ala Leu
Ala Pro Ala Ala Leu Ala Leu Leu Pro 1 5 10
2912PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 29Ala
Leu Ile Ala Ala Pro Ile Leu Ala Leu Ala Pro 1 5
10 3012PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 30Ala Val Val Ala Ala Pro Leu Val Leu Ala Leu Pro 1
5 10 3112PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 31Leu Leu Ala Leu Ala Pro Ala Ala Leu Leu Ala Pro 1
5 10 3212PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 32Ala Ile Val Ala Leu Pro Ala Leu
Ala Leu Ala Pro 1 5 10
3312PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 33Ala Ala Ile Ile
Val Pro Ala Ala Leu Leu Ala Pro 1 5 10
3412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 34Ile Ala Val
Ala Leu Pro Ala Leu Ile Ala Ala Pro 1 5 10
3512PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 35Ala
Val Ile Val Leu Pro Ala Leu Ala Val Ala Pro 1 5
10 3612PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 36Ala Val Leu Ala Val Pro Ala Val Leu Val Ala Pro 1
5 10 3712PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 37Val Leu Ala Ile Val Pro Ala Val Ala Leu Ala Pro 1
5 10 3812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 38Leu Leu Ala Val Val Pro Ala Val
Ala Leu Ala Pro 1 5 10
3912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 39Ala Val Ile Ala
Leu Pro Ala Leu Ile Ala Ala Pro 1 5 10
4012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 40Ala Val Val
Ala Leu Pro Ala Ala Leu Ile Val Pro 1 5 10
4112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 41Leu
Ala Leu Val Leu Pro Ala Ala Leu Ala Ala Pro 1 5
10 4212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 42Leu Ala Ala Val Leu Pro Ala Leu Leu Ala Ala Pro 1
5 10 4312PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 43Ala Leu Ala Val Pro Val Ala Leu Ala Ile Val Pro 1
5 10 4412PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 44Ala Leu Ile Ala Pro Val Val Ala
Leu Val Ala Pro 1 5 10
4512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 45Leu Leu Ala Ala
Pro Val Val Ile Ala Leu Ala Pro 1 5 10
4612PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 46Leu Ala Ala
Ile Val Pro Ala Ile Ile Ala Val Pro 1 5 10
4712PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 47Ala
Ala Leu Val Leu Pro Leu Ile Ile Ala Ala Pro 1 5
10 4812PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 48Leu Ala Leu Ala Val Pro Ala Leu Ala Ala Leu Pro 1
5 10 4912PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 49Leu Ile Ala Ala Leu Pro Ala Val Ala Ala Leu Pro 1
5 10 5012PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 50Ala Leu Ala Leu Val Pro Ala Ile
Ala Ala Leu Pro 1 5 10
5112PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 51Ala Ala Ile Leu
Ala Pro Ile Val Ala Leu Ala Pro 1 5 10
5212PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 52Ala Leu Leu
Ile Ala Pro Ala Ala Val Ile Ala Pro 1 5 10
5312PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 53Ala
Ile Leu Ala Val Pro Ile Ala Val Val Ala Pro 1 5
10 5412PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 54Ile Leu Ala Ala Val Pro Ile Ala Leu Ala Ala Pro 1
5 10 5512PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 55Val Ala Ala Leu Leu Pro Ala Ala Ala Val Leu Pro 1
5 10 5612PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 56Ala Ala Ala Val Val Pro Val Leu
Leu Val Ala Pro 1 5 10
5712PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 57Ala Ala Leu Leu
Val Pro Ala Leu Val Ala Ala Pro 1 5 10
5812PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 58Ala Ala Val
Leu Leu Pro Val Ala Leu Ala Ala Pro 1 5 10
5912PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 59Ala
Ala Ala Leu Ala Pro Val Leu Ala Leu Val Pro 1 5
10 6012PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 60Leu Val Leu Val Pro Leu Leu Ala Ala Ala Ala Pro 1
5 10 6112PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 61Ala Leu Ile Ala Val Pro Ala Ile Ile Val Ala Pro 1
5 10 6212PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 62Ala Leu Ala Val Ile Pro Ala Ala
Ala Ile Leu Pro 1 5 10
6312PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 63Leu Ala Ala Ala
Pro Val Val Ile Val Ile Ala Pro 1 5 10
6412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 64Val Leu Ala
Ile Ala Pro Leu Leu Ala Ala Val Pro 1 5 10
6512PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 65Ala
Leu Ile Val Leu Pro Ala Ala Val Ala Val Pro 1 5
10 6612PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 66Val Leu Ala Val Ala Pro Ala Leu Ile Val Ala Pro 1
5 10 6712PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 67Ala Ala Leu Leu Ala Pro Ala Leu Ile Val Ala Pro 1
5 10 6812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 68Ala Leu Ile Ala Pro Ala Val Ala
Leu Ile Val Pro 1 5 10
6912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 69Ala Ile Val Leu
Leu Pro Ala Ala Val Val Ala Pro 1 5 10
7012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 70Val Ile Ala
Ala Pro Val Leu Ala Val Leu Ala Pro 1 5 10
7112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 71Leu
Ala Leu Ala Pro Ala Leu Ala Leu Leu Ala Pro 1 5
10 7212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 72Ala Ile Ile Leu Ala Pro Ile Ala Ala Ile Ala Pro 1
5 10 7312PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 73Ile Ala Leu Ala Ala Pro Ile Leu Leu Ala Ala Pro 1
5 10 7412PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 74Ile Val Ala Val Ala Leu Pro Ala
Leu Ala Val Pro 1 5 10
7512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 75Val Val Ala Ile
Val Leu Pro Ala Leu Ala Ala Pro 1 5 10
7612PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 76Ile Val Ala
Val Ala Leu Pro Val Ala Leu Ala Pro 1 5 10
7712PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 77Ile
Val Ala Val Ala Leu Pro Ala Ala Leu Val Pro 1 5
10 7812PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 78Ile Val Ala Val Ala Leu Pro Ala Val Ala Leu Pro 1
5 10 7912PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 79Ile Val Ala Val Ala Leu Pro Ala Val Leu Ala Pro 1
5 10 8012PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 80Val Ile Val Ala Leu Ala Pro Ala
Val Leu Ala Pro 1 5 10
8112PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 81Ile Val Ala Val
Ala Leu Pro Ala Leu Val Ala Pro 1 5 10
8212PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 82Ala Leu Leu
Ile Val Ala Pro Val Ala Val Ala Pro 1 5 10
8312PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 83Ala
Val Val Ile Val Ala Pro Ala Val Ile Ala Pro 1 5
10 8412PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 84Ala Val Leu Ala Val Ala Pro Ala Leu Ile Val Pro 1
5 10 8512PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 85Leu Val Ala Ala Val Ala Pro Ala Leu Ile Val Pro 1
5 10 8612PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 86Ala Val Ile Val Val Ala Pro Ala
Leu Leu Ala Pro 1 5 10
8712PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 87Val Val Ala Ile
Val Leu Pro Ala Val Ala Ala Pro 1 5 10
8812PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 88Ala Ala Ala
Leu Val Ile Pro Ala Ile Leu Ala Pro 1 5 10
8912PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 89Val
Ile Val Ala Leu Ala Pro Ala Leu Leu Ala Pro 1 5
10 9012PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 90Val Ile Val Ala Ile Ala Pro Ala Leu Leu Ala Pro 1
5 10 9112PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 91Ile Val Ala Ile Ala Val Pro Ala Leu Val Ala Pro 1
5 10 9212PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 92Ala Ala Leu Ala Val Ile Pro Ala
Ala Ile Leu Pro 1 5 10
9312PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 93Ala Leu Ala Ala
Val Ile Pro Ala Ala Ile Leu Pro 1 5 10
9412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 94Ala Ala Ala
Leu Val Ile Pro Ala Ala Ile Leu Pro 1 5 10
9512PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 95Leu
Ala Ala Ala Val Ile Pro Ala Ala Ile Leu Pro 1 5
10 9612PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 96Leu Ala Ala Ala Val Ile Pro Val Ala Ile Leu Pro 1
5 10 9712PRTArtificial SequenceAdvanced
Macromolecule Transduction Domain (aMTD) Sequences for Improvement
of Cell-Permeability 97Ala Ala Ile Leu Ala Ala Pro Leu Ile Ala Val Pro 1
5 10 9812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 98Val Val Ala Ile Leu Ala Pro Leu
Leu Ala Ala Pro 1 5 10
9912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 99Ala Val Val Val
Ala Ala Pro Val Leu Ala Leu Pro 1 5 10
10012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 100Ala Val Val
Ala Ile Ala Pro Val Leu Ala Leu Pro 1 5 10
10112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 101Ala
Leu Ala Ala Leu Val Pro Ala Val Leu Val Pro 1 5
10 10212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 102Ala Leu Ala Ala Leu Val Pro Val Ala Leu Val Pro 1
5 10 10312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 103Leu Ala Ala Ala Leu Val Pro Val
Ala Leu Val Pro 1 5 10
10412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 104Ala Leu Ala Ala
Leu Val Pro Ala Leu Val Val Pro 1 5 10
10512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 105Ile Ala Ala
Val Ile Val Pro Ala Val Ala Leu Pro 1 5 10
10612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 106Ile
Ala Ala Val Leu Val Pro Ala Val Ala Leu Pro 1 5
10 10712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 107Ala Val Ala Ile Leu Val Pro Leu Leu Ala Ala Pro 1
5 10 10812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 108Ala Val Val Ile Leu Val Pro Leu
Ala Ala Ala Pro 1 5 10
10912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 109Ile Ala Ala Val
Ile Val Pro Val Ala Ala Leu Pro 1 5 10
11012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 110Ala Ile Ala
Ile Ala Ile Val Pro Val Ala Leu Pro 1 5 10
11112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 111Ile
Leu Ala Val Ala Ala Ile Pro Val Ala Val Pro 1 5
10 11212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 112Ile Leu Ala Ala Ala Ile Ile Pro Ala Ala Leu Pro 1
5 10 11312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 113Leu Ala Val Val Leu Ala Ala Pro
Ala Ile Val Pro 1 5 10
11412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 114Ala Ile Leu Ala
Ala Ile Val Pro Leu Ala Val Pro 1 5 10
11512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 115Val Ile Val
Ala Leu Ala Val Pro Ala Leu Ala Pro 1 5 10
11612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 116Ala
Ile Val Ala Leu Ala Val Pro Val Leu Ala Pro 1 5
10 11712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 117Ala Ala Ile Ile Ile Val Leu Pro Ala Ala Leu Pro 1
5 10 11812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 118Leu Ile Val Ala Leu Ala Val Pro
Ala Leu Ala Pro 1 5 10
11912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 119Ala Ile Ile Ile
Val Ile Ala Pro Ala Ala Ala Pro 1 5 10
12012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 120Leu Ala Ala
Leu Ile Val Val Pro Ala Val Ala Pro 1 5 10
12112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 121Ala
Leu Leu Val Ile Ala Val Pro Ala Val Ala Pro 1 5
10 12212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 122Ala Val Ala Leu Ile Val Val Pro Ala Leu Ala Pro 1
5 10 12312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 123Ala Leu Ala Ile Val Val Ala Pro
Val Ala Val Pro 1 5 10
12412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 124Leu Leu Ala Leu
Ile Ile Ala Pro Ala Ala Ala Pro 1 5 10
12512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 125Ala Leu Ala
Leu Ile Ile Val Pro Ala Val Ala Pro 1 5 10
12612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 126Leu
Leu Ala Ala Leu Ile Ala Pro Ala Ala Leu Pro 1 5
10 12712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 127Ile Val Ala Leu Ile Val Ala Pro Ala Ala Val Pro 1
5 10 12812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 128Val Val Leu Val Leu Ala Ala Pro
Ala Ala Val Pro 1 5 10
12912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 129Ala Ala Val Ala
Ile Val Leu Pro Ala Val Val Pro 1 5 10
13012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 130Ala Leu Ile
Ala Ala Ile Val Pro Ala Leu Val Pro 1 5 10
13112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 131Ala
Leu Ala Val Ile Val Val Pro Ala Leu Ala Pro 1 5
10 13212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 132Val Ala Ile Ala Leu Ile Val Pro Ala Leu Ala Pro 1
5 10 13312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 133Val Ala Ile Val Leu Val Ala Pro
Ala Val Ala Pro 1 5 10
13412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 134Val Ala Val Ala
Leu Ile Val Pro Ala Leu Ala Pro 1 5 10
13512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 135Ala Val Ile
Leu Ala Leu Ala Pro Ile Val Ala Pro 1 5 10
13612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 136Ala
Leu Ile Val Ala Ile Ala Pro Ala Leu Val Pro 1 5
10 13712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 137Ala Ala Ile Leu Ile Ala Val Pro Ile Ala Ala Pro 1
5 10 13812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 138Val Ile Val Ala Leu Ala Ala Pro
Val Leu Ala Pro 1 5 10
13912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 139Val Leu Val Ala
Leu Ala Ala Pro Val Ile Ala Pro 1 5 10
14012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 140Val Ala Leu
Ile Ala Val Ala Pro Ala Val Val Pro 1 5 10
14112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 141Val
Ile Ala Ala Val Leu Ala Pro Val Ala Val Pro 1 5
10 14212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 142Ala Leu Ile Val Leu Ala Ala Pro Val Ala Val Pro 1
5 10 14312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 143Val Ala Ala Ala Ile Ala Leu Pro
Ala Ile Val Pro 1 5 10
14412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 144Ile Leu Ala Ala
Ala Ala Ala Pro Leu Ile Val Pro 1 5 10
14512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 145Leu Ala Leu
Val Leu Ala Ala Pro Ala Ile Val Pro 1 5 10
14612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 146Ala
Leu Ala Val Val Ala Leu Pro Ala Ile Val Pro 1 5
10 14712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 147Ala Ala Ile Leu Ala Pro Ile Val Ala Ala Leu Pro 1
5 10 14812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 148Ile Leu Ile Ala Ile Ala Ile Pro
Ala Ala Ala Pro 1 5 10
14912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 149Leu Ala Ile Val
Leu Ala Ala Pro Val Ala Val Pro 1 5 10
15012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 150Ala Ala Ile
Ala Ile Ile Ala Pro Ala Ile Val Pro 1 5 10
15112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 151Leu
Ala Val Ala Ile Val Ala Pro Ala Leu Val Pro 1 5
10 15212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 152Leu Ala Ile Val Leu Ala Ala Pro Ala Val Leu Pro 1
5 10 15312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 153Ala Ala Ile Val Leu Ala Leu Pro
Ala Val Leu Pro 1 5 10
15412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 154Ala Leu Leu Val
Ala Val Leu Pro Ala Ala Leu Pro 1 5 10
15512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 155Ala Ala Leu
Val Ala Val Leu Pro Val Ala Leu Pro 1 5 10
15612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 156Ala
Ile Leu Ala Val Ala Leu Pro Leu Leu Ala Pro 1 5
10 15712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 157Ile Val Ala Val Ala Leu Val Pro Ala Leu Ala Pro 1
5 10 15812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 158Ile Val Ala Val Ala Leu Leu Pro
Ala Leu Ala Pro 1 5 10
15912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 159Ile Val Ala Val
Ala Leu Leu Pro Ala Val Ala Pro 1 5 10
16012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 160Ile Val Ala
Leu Ala Val Leu Pro Ala Val Ala Pro 1 5 10
16112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 161Val
Ala Val Leu Ala Val Leu Pro Ala Leu Ala Pro 1 5
10 16212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 162Ile Ala Val Leu Ala Val Ala Pro Ala Val Leu Pro 1
5 10 16312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 163Leu Ala Val Ala Ile Ile Ala Pro
Ala Val Ala Pro 1 5 10
16412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 164Val Ala Leu Ala
Ile Ala Leu Pro Ala Val Leu Pro 1 5 10
16512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 165Ala Ile Ala
Ile Ala Leu Val Pro Val Ala Leu Pro 1 5 10
16612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 166Ala
Ala Val Val Ile Val Ala Pro Val Ala Leu Pro 1 5
10 16712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 167Val Ala Ile Ile Val Val Ala Pro Ala Leu Ala Pro 1
5 10 16812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 168Val Ala Leu Leu Ala Ile Ala Pro
Ala Leu Ala Pro 1 5 10
16912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 169Val Ala Val Leu
Ile Ala Val Pro Ala Leu Ala Pro 1 5 10
17012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 170Ala Val Ala
Leu Ala Val Leu Pro Ala Val Val Pro 1 5 10
17112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 171Ala
Val Ala Leu Ala Val Val Pro Ala Val Leu Pro 1 5
10 17212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 172Ile Val Val Ile Ala Val Ala Pro Ala Val Ala Pro 1
5 10 17312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 173Ile Val Val Ala Ala Val Val Pro
Ala Leu Ala Pro 1 5 10
17412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 174Ile Val Ala Leu
Val Pro Ala Val Ala Ile Ala Pro 1 5 10
17512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 175Val Ala Ala
Leu Pro Ala Val Ala Leu Val Val Pro 1 5 10
17612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 176Leu
Val Ala Ile Ala Pro Leu Ala Val Leu Ala Pro 1 5
10 17712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 177Ala Val Ala Leu Val Pro Val Ile Val Ala Ala Pro 1
5 10 17812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 178Ala Ile Ala Val Ala Ile Ala Pro
Val Ala Leu Pro 1 5 10
17912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 179Ala Ile Ala Leu
Ala Val Pro Val Leu Ala Leu Pro 1 5 10
18012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 180Leu Val Leu
Ile Ala Ala Ala Pro Ile Ala Leu Pro 1 5 10
18112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 181Leu
Val Ala Leu Ala Val Pro Ala Ala Val Leu Pro 1 5
10 18212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 182Ala Val Ala Leu Ala Val Pro Ala Leu Val Leu Pro 1
5 10 18312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 183Leu Val Val Leu Ala Ala Ala Pro
Leu Ala Val Pro 1 5 10
18412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 184Leu Ile Val Leu
Ala Ala Pro Ala Leu Ala Ala Pro 1 5 10
18512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 185Val Ile Val
Leu Ala Ala Pro Ala Leu Ala Ala Pro 1 5 10
18612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 186Ala
Val Val Leu Ala Val Pro Ala Leu Ala Val Pro 1 5
10 18712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 187Leu Ile Ile Val Ala Ala Ala Pro Ala Val Ala Pro 1
5 10 18812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 188Ile Val Ala Val Ile Val Ala Pro
Ala Val Ala Pro 1 5 10
18912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 189Leu Val Ala Leu
Ala Ala Pro Ile Ile Ala Val Pro 1 5 10
19012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 190Ile Ala Ala
Val Leu Ala Ala Pro Ala Leu Val Pro 1 5 10
19112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 191Ile
Ala Leu Leu Ala Ala Pro Ile Ile Ala Val Pro 1 5
10 19212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 192Ala Ala Leu Ala Leu Val Ala Pro Val Ile Val Pro 1
5 10 19312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 193Ile Ala Leu Val Ala Ala Pro Val
Ala Leu Val Pro 1 5 10
19412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 194Ile Ile Val Ala
Val Ala Pro Ala Ala Ile Val Pro 1 5 10
19512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 195Ala Val Ala
Ala Ile Val Pro Val Ile Val Ala Pro 1 5 10
19612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 196Ala
Val Leu Val Leu Val Ala Pro Ala Ala Ala Pro 1 5
10 19712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 197Val Val Ala Leu Leu Ala Pro Leu Ile Ala Ala Pro 1
5 10 19812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 198Ala Ala Val Val Ile Ala Pro Leu
Leu Ala Val Pro 1 5 10
19912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 199Ile Ala Val Ala
Val Ala Ala Pro Leu Leu Val Pro 1 5 10
20012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 200Leu Val Ala
Ile Val Val Leu Pro Ala Val Ala Pro 1 5 10
20112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 201Ala
Val Ala Ile Val Val Leu Pro Ala Val Ala Pro 1 5
10 20212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 202Ala Val Ile Leu Leu Ala Pro Leu Ile Ala Ala Pro 1
5 10 20312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 203Leu Val Ile Ala Leu Ala Ala Pro
Val Ala Leu Pro 1 5 10
20412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 204Val Leu Ala Val
Val Leu Pro Ala Val Ala Leu Pro 1 5 10
20512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 205Val Leu Ala
Val Ala Ala Pro Ala Val Leu Leu Pro 1 5 10
20612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 206Ala
Ala Val Val Leu Leu Pro Ile Ile Ala Ala Pro 1 5
10 20712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 207Ala Leu Leu Val Ile Ala Pro Ala Ile Ala Val Pro 1
5 10 20812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 208Ala Val Leu Val Ile Ala Val Pro
Ala Ile Ala Pro 1 5 10
20912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 209Ala Leu Leu Val
Val Ile Ala Pro Leu Ala Ala Pro 1 5 10
21012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 210Val Leu Val
Ala Ala Ile Leu Pro Ala Ala Ile Pro 1 5 10
21112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 211Val
Leu Val Ala Ala Val Leu Pro Ile Ala Ala Pro 1 5
10 21212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 212Val Leu Ala Ala Ala Val Leu Pro Leu Val Val Pro 1
5 10 21312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 213Ala Ile Ala Ile Val Val Pro Ala
Val Ala Val Pro 1 5 10
21412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 214Val Ala Ile Ile
Ala Val Pro Ala Val Val Ala Pro 1 5 10
21512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 215Ile Val Ala
Leu Val Ala Pro Ala Ala Val Val Pro 1 5 10
21612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 216Ala
Ala Ile Val Leu Leu Pro Ala Val Val Val Pro 1 5
10 21712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 217Ala Ala Leu Ile Val Val Pro Ala Val Ala Val Pro 1
5 10 21812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 218Ala Ile Ala Leu Val Val Pro Ala
Val Ala Val Pro 1 5 10
21912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 219Leu Ala Ile Val
Pro Ala Ala Ile Ala Ala Leu Pro 1 5 10
22012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 220Leu Val Ala
Ile Ala Pro Ala Val Ala Val Leu Pro 1 5 10
22112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 221Val
Leu Ala Val Ala Pro Ala Val Ala Val Leu Pro 1 5
10 22212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 222Ile Leu Ala Val Val Ala Ile Pro Ala Ala Ala Pro 1
5 10 22312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 223Ile Leu Val Ala Ala Ala Pro Ile
Ala Ala Leu Pro 1 5 10
22412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 224Ile Leu Ala Val
Ala Ala Ile Pro Ala Ala Leu Pro 1 5 10
22512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 225Val Ile Ala
Ile Pro Ala Ile Leu Ala Ala Ala Pro 1 5 10
22612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 226Ala
Ile Ile Ile Val Val Pro Ala Ile Ala Ala Pro 1 5
10 22712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 227Ala Ile Leu Ile Val Val Ala Pro Ile Ala Ala Pro 1
5 10 22812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 228Ala Val Ile Val Pro Val Ala Ile
Ile Ala Ala Pro 1 5 10
22912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 229Ala Val Val Ile
Ala Leu Pro Ala Val Val Ala Pro 1 5 10
23012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 230Ala Leu Val
Ala Val Ile Ala Pro Val Val Ala Pro 1 5 10
23112PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 231Ala
Leu Val Ala Val Leu Pro Ala Val Ala Val Pro 1 5
10 23212PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 232Ala Leu Val Ala Pro Leu Leu Ala Val Ala Val Pro 1
5 10 23312PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 233Ala Val Leu Ala Val Val Ala Pro
Val Val Ala Pro 1 5 10
23412PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 234Ala Val Ile Ala
Val Ala Pro Leu Val Val Ala Pro 1 5 10
23512PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 235Ala Val Ile
Ala Leu Ala Pro Val Val Val Ala Pro 1 5 10
23612PRTArtificial SequenceAdvanced Macromolecule Transduction
Domain (aMTD) Sequences for Improvement of Cell-Permeability 236Val
Ala Ile Ala Leu Ala Pro Val Val Val Ala Pro 1 5
10 23712PRTArtificial SequenceAdvanced Macromolecule
Transduction Domain (aMTD) Sequences for Improvement of
Cell-Permeability 237Val Ala Leu Ala Leu Ala Pro Val Val Val Ala Pro 1
5 10 23812PRTArtificial
SequenceAdvanced Macromolecule Transduction Domain (aMTD) Sequences
for Improvement of Cell-Permeability 238Val Ala Ala Leu Leu Pro Ala Val
Val Val Ala Pro 1 5 10
23912PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 239Val Ala Leu Ala
Leu Pro Ala Val Val Val Ala Pro 1 5 10
24012PRTArtificial SequenceAdvanced Macromolecule Transduction Domain
(aMTD) Sequences for Improvement of Cell-Permeability 240Val Ala Leu
Leu Ala Pro Ala Val Val Val Ala Pro 1 5 10
24136DNAArtificial SequenceThe polynucleotide sequence that
encodes advanced Macromolecule Transduction Domain (aMTD) peptide
for Improvement of Cell-Permeability 241gcggcggcgc tggcgccggt
ggtgctggcg ctgccg 3624236DNAArtificial
SequenceThe polynucleotide sequence that encodes advanced
Macromolecule Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 242gcggcggcgg tgccgctgct ggcggtggtg gtgccg
3624336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 243gcggcgctgc
tggtgccggc ggcggtgctg gcgccg
3624436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 244gcgctggcgc tgctgccggt ggcggcgctg
gcgccg 3624536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 245gcggcggcgc tgctgccggt ggcgctggtg gcgccg
3624636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 246gtggtggcgc
tggcgccggc gctggcggcg ctgccg
3624736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 247ctgctggcgg cggtgccggc ggtgctgctg
gcgccg 3624836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 248gcggcggcgc tggtgccggt ggtggcgctg ctgccg
3624936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 249gcggtggcgc
tgctgccggc gctgctggcg gtgccg
3625036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 250gcggtggtgc tggtgccggt gctggcggcg
gcgccg 3625136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 251gtggtgctgg tgctgccggc ggcggcggcg gtgccg
3625236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 252attgcgctgg
cggcgccggc gctgattgtg gcgccg
3625336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 253attgtggcgg tggcgccggc gctggtggcg
ctgccg 3625436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 254gtggcggcgc tgccggtggt ggcggtggtg gcgccg
3625536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 255ctgctggcgg
cgccgctggt ggtggcggcg gtgccg
3625636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 256gcgctggcgg tgccggtggc gctgctggtg
gcgccg 3625736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 257gtggcggcgc tgccggtgct gctggcggcg ctgccg
3625836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 258gtggcgctgc
tggcgccggt ggcgctggcg gtgccg
3625936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 259gcggcgctgc tggtgccggc gctggtggcg
gtgccg 3626036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 260gcgattgtgg cgctgccggt ggcggtgctg gcgccg
3626136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 261attgcgattg
tggcgccggt ggtggcgctg gcgccg
3626236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 262gcggcgctgc tgccggcgct ggcggcgctg
ctgccg 3626336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 263gcggtggtgc tggcgccggt ggcggcggtg ctgccg
3626436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 264ctggcggtgg
cggcgccgct ggcgctggcg ctgccg
3626536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 265gcggcggtgg cggcgccgct gctgctggcg
ctgccg 3626636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 266ctgctggtgc tgccggcggc ggcgctggcg gcgccg
3626736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 267ctggtggcgg
tggcgccggt ggcggcggtg ctgccg
3626836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 268ctggcgctgg cgccggcggc gctggcgctg
ctgccg 3626936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 269gcgctgattg cggcgccgat tctggcgctg gcgccg
3627036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 270gcggtggtgg
cggcgccgct ggtgctggcg ctgccg
3627136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 271ctgctggcgc tggcgccggc ggcgctgctg
gcgccg 3627236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 272gcgattgtgg cgctgccggc gctggcgctg gcgccg
3627336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 273gcggcgatta
ttgtgccggc ggcgctgctg gcgccg
3627436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 274attgcggtgg cgctgccggc gctgattgcg
gcgccg 3627536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 275gcggtgattg tgctgccggc gctggcggtg gcgccg
3627636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 276gcggtgctgg
cggtgccggc ggtgctggtg gcgccg
3627736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 277gtgctggcga ttgtgccggc ggtggcgctg
gcgccg 3627836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 278ctgctggcgg tggtgccggc ggtggcgctg gcgccg
3627936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 279gcggtgattg
cgctgccggc gctgattgcg gcgccg
3628036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 280gcggtggtgg cgctgccggc ggcgctgatt
gtgccg 3628136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 281ctggcgctgg tgctgccggc ggcgctggcg gcgccg
3628236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 282ctggcggcgg
tgctgccggc gctgctggcg gcgccg
3628336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 283gcgctggcgg tgccggtggc gctggcgatt
gtgccg 3628436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 284gcgctgattg cgccggtggt ggcgctggtg gcgccg
3628536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 285ctgctggcgg
cgccggtggt gattgcgctg gcgccg
3628636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 286ctggcggcga ttgtgccggc gattattgcg
gtgccg 3628736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 287gcggcgctgg tgctgccgct gattattgcg gcgccg
3628836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 288ctggcgctgg
cggtgccggc gctggcggcg ctgccg
3628936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 289ctgattgcgg cgctgccggc ggtggcggcg
ctgccg 3629036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 290gcgctggcgc tggtgccggc gattgcggcg ctgccg
3629136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 291gcggcgattc
tggcgccgat tgtggcgctg gcgccg
3629236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 292gcgctgctga ttgcgccggc ggcggtgatt
gcgccg 3629336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 293gcgattctgg cggtgccgat tgcggtggtg gcgccg
3629436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 294attctggcgg
cggtgccgat tgcgctggcg gcgccg
3629536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 295gtggcggcgc tgctgccggc ggcggcggtg
ctgccg 3629636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 296gcggcggcgg tggtgccggt gctgctggtg gcgccg
3629736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 297gcggcgctgc
tggtgccggc gctggtggcg gcgccg
3629836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 298gcggcggtgc tgctgccggt ggcgctggcg
gcgccg 3629936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 299gcggcggcgc tggcgccggt gctggcgctg gtgccg
3630036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 300ctggtgctgg
tgccgctgct ggcggcggcg gcgccg
3630136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 301gcgctgattg cggtgccggc gattattgtg
gcgccg 3630236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 302gcgctggcgg tgattccggc ggcggcgatt ctgccg
3630336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 303ctggcggcgg
cgccggtggt gattgtgatt gcgccg
3630436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 304gtgctggcga ttgcgccgct gctggcggcg
gtgccg 3630536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 305gcgctgattg tgctgccggc ggcggtggcg gtgccg
3630636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 306gtgctggcgg
tggcgccggc gctgattgtg gcgccg
3630736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 307gcggcgctgc tggcgccggc gctgattgtg
gcgccg 3630836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 308gcgctgattg cgccggcggt ggcgctgatt gtgccg
3630936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 309gcgattgtgc
tgctgccggc ggcggtggtg gcgccg
3631036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 310gtgattgcgg cgccggtgct ggcggtgctg
gcgccg 3631136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 311ctggcgctgg cgccggcgct ggcgctgctg gcgccg
3631236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 312gcgattattc
tggcgccgat tgcggcgatt gcgccg
3631336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 313attgcgctgg cggcgccgat tctgctggcg
gcgccg 3631436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 314attgtggcgg tggcgctgcc ggcgctggcg gtgccg
3631536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 315gtggtggcga
ttgtgctgcc ggcgctggcg gcgccg
3631636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 316attgtggcgg tggcgctgcc ggtggcgctg
gcgccg 3631736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 317attgtggcgg tggcgctgcc ggcggcgctg gtgccg
3631836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 318attgtggcgg
tggcgctgcc ggcggtggcg ctgccg
3631936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 319attgtggcgg tggcgctgcc ggcggtgctg
gcgccg 3632036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 320gtgattgtgg cgctggcgcc ggcggtgctg gcgccg
3632136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 321attgtggcgg
tggcgctgcc ggcgctggtg gcgccg
3632236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 322gcgctgctga ttgtggcgcc ggtggcggtg
gcgccg 3632336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 323gcggtggtga ttgtggcgcc ggcggtgatt gcgccg
3632436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 324gcggtgctgg
cggtggcgcc ggcgctgatt gtgccg
3632536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 325ctggtggcgg cggtggcgcc ggcgctgatt
gtgccg 3632636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 326gcggtgattg tggtggcgcc ggcgctgctg gcgccg
3632736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 327gtggtggcga
ttgtgctgcc ggcggtggcg gcgccg
3632836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 328gcggcggcgc tggtgattcc ggcgattctg
gcgccg 3632936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 329gtgattgtgg cgctggcgcc ggcgctgctg gcgccg
3633036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 330gtgattgtgg
cgattgcgcc ggcgctgctg gcgccg
3633136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 331attgtggcga ttgcggtgcc ggcgctggtg
gcgccg 3633236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 332gcggcgctgg cggtgattcc ggcggcgatt ctgccg
3633336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 333gcgctggcgg
cggtgattcc ggcggcgatt ctgccg
3633436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 334gcggcggcgc tggtgattcc ggcggcgatt
ctgccg 3633536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 335ctggcggcgg cggtgattcc ggcggcgatt ctgccg
3633636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 336ctggcggcgg
cggtgattcc ggtggcgatt ctgccg
3633736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 337gcggcgattc tggcggcgcc gctgattgcg
gtgccg 3633836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 338gtggtggcga ttctggcgcc gctgctggcg gcgccg
3633936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 339gcggtggtgg
tggcggcgcc ggtgctggcg ctgccg
3634036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 340gcggtggtgg cgattgcgcc ggtgctggcg
ctgccg 3634136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 341gcgctggcgg cgctggtgcc ggcggtgctg gtgccg
3634236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 342gcgctggcgg
cgctggtgcc ggtggcgctg gtgccg
3634336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 343ctggcggcgg cgctggtgcc ggtggcgctg
gtgccg 3634436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 344gcgctggcgg cgctggtgcc ggcgctggtg gtgccg
3634536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 345attgcggcgg
tgattgtgcc ggcggtggcg ctgccg
3634636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 346attgcggcgg tgctggtgcc ggcggtggcg
ctgccg 3634736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 347gcggtggcga ttctggtgcc gctgctggcg gcgccg
3634836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 348gcggtggtga
ttctggtgcc gctggcggcg gcgccg
3634936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 349attgcggcgg tgattgtgcc ggtggcggcg
ctgccg 3635036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 350gcgattgcga ttgcgattgt gccggtggcg ctgccg
3635136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 351attctggcgg
tggcggcgat tccggtggcg gtgccg
3635236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 352attctggcgg cggcgattat tccggcggcg
ctgccg 3635336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 353ctggcggtgg tgctggcggc gccggcgatt gtgccg
3635436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 354gcgattctgg
cggcgattgt gccgctggcg gtgccg
3635536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 355gtgattgtgg cgctggcggt gccggcgctg
gcgccg 3635636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 356gcgattgtgg cgctggcggt gccggtgctg gcgccg
3635736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 357gcggcgatta
ttattgtgct gccggcggcg ctgccg
3635836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 358ctgattgtgg cgctggcggt gccggcgctg
gcgccg 3635936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 359gcgattatta ttgtgattgc gccggcggcg gcgccg
3636036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 360ctggcggcgc
tgattgtggt gccggcggtg gcgccg
3636136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 361gcgctgctgg tgattgcggt gccggcggtg
gcgccg 3636236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 362gcggtggcgc tgattgtggt gccggcgctg gcgccg
3636336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 363gcgctggcga
ttgtggtggc gccggtggcg gtgccg
3636436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 364ctgctggcgc tgattattgc gccggcggcg
gcgccg 3636536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 365gcgctggcgc tgattattgt gccggcggtg gcgccg
3636636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 366ctgctggcgg
cgctgattgc gccggcggcg ctgccg
3636736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 367attgtggcgc tgattgtggc gccggcggcg
gtgccg 3636836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 368gtggtgctgg tgctggcggc gccggcggcg gtgccg
3636936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 369gcggcggtgg
cgattgtgct gccggcggtg gtgccg
3637036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 370gcgctgattg cggcgattgt gccggcgctg
gtgccg 3637136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 371gcgctggcgg tgattgtggt gccggcgctg gcgccg
3637236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 372gtggcgattg
cgctgattgt gccggcgctg gcgccg
3637336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 373gtggcgattg tgctggtggc gccggcggtg
gcgccg 3637436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 374gtggcggtgg cgctgattgt gccggcgctg gcgccg
3637536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 375gcggtgattc
tggcgctggc gccgattgtg gcgccg
3637636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 376gcgctgattg tggcgattgc gccggcgctg
gtgccg 3637736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 377gcggcgattc tgattgcggt gccgattgcg gcgccg
3637836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 378gtgattgtgg
cgctggcggc gccggtgctg gcgccg
3637936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 379gtgctggtgg cgctggcggc gccggtgatt
gcgccg 3638036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 380gtggcgctga ttgcggtggc gccggcggtg gtgccg
3638136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 381gtgattgcgg
cggtgctggc gccggtggcg gtgccg
3638236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 382gcgctgattg tgctggcggc gccggtggcg
gtgccg 3638336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 383gtggcggcgg cgattgcgct gccggcgatt gtgccg
3638436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 384attctggcgg
cggcggcggc gccgctgatt gtgccg
3638536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 385ctggcgctgg tgctggcggc gccggcgatt
gtgccg 3638636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 386gcgctggcgg tggtggcgct gccggcgatt gtgccg
3638736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 387gcggcgattc
tggcgccgat tgtggcggcg ctgccg
3638836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 388attctgattg cgattgcgat tccggcggcg
gcgccg 3638936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 389ctggcgattg tgctggcggc gccggtggcg gtgccg
3639036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 390gcggcgattg
cgattattgc gccggcgatt gtgccg
3639136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 391ctggcggtgg cgattgtggc gccggcgctg
gtgccg 3639236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 392ctggcgattg tgctggcggc gccggcggtg ctgccg
3639336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 393gcggcgattg
tgctggcgct gccggcggtg ctgccg
3639436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 394gcgctgctgg tggcggtgct gccggcggcg
ctgccg 3639536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 395gcggcgctgg tggcggtgct gccggtggcg ctgccg
3639636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 396gcgattctgg
cggtggcgct gccgctgctg gcgccg
3639736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 397attgtggcgg tggcgctggt gccggcgctg
gcgccg 3639836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 398attgtggcgg tggcgctgct gccggcgctg gcgccg
3639936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 399attgtggcgg
tggcgctgct gccggcggtg gcgccg
3640036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 400attgtggcgc tggcggtgct gccggcggtg
gcgccg 3640136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 401gtggcggtgc tggcggtgct gccggcgctg gcgccg
3640236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 402attgcggtgc
tggcggtggc gccggcggtg ctgccg
3640336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 403ctggcggtgg cgattattgc gccggcggtg
gcgccg 3640436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 404gtggcgctgg cgattgcgct gccggcggtg ctgccg
3640536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 405gcgattgcga
ttgcgctggt gccggtggcg ctgccg
3640636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 406gcggcggtgg tgattgtggc gccggtggcg
ctgccg 3640736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 407gcggcgattc tggcgattgt ggcgccgctg gcgccg
3640836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 408gtggcgctgc
tggcgattgc gccggcgctg gcgccg
3640936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 409gtggcggtgc tgattgcggt gccggcgctg
gcgccg 3641036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 410gcggtggcgc tggcggtgct gccggcggtg gtgccg
3641136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 411gcggtggcgc
tggcggtggt gccggcggtg ctgccg
3641236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 412attgtggtga ttgcggtggc gccggcggtg
gcgccg 3641336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 413attgtggtgg cggcggtggt gccggcgctg gcgccg
3641436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 414attgtggcgc
tggtgccggc ggtggcgatt gcgccg
3641536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 415gtggcggcgc tgccggcggt ggcgctggtg
gtgccg 3641636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 416ctggtggcga ttgcgccgct ggcggtgctg gcgccg
3641736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 417gcggtggcgc
tggtgccggt gattgtggcg gcgccg
3641836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 418gcgattgcgg tggcgattgc gccggtggcg
ctgccg 3641936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 419gcgattgcgc tggcggtgcc ggtgctggcg ctgccg
3642036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 420ctggtgctga
ttgcggcggc gccgattgcg ctgccg
3642136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 421ctggtggcgc tggcggtgcc ggcggcggtg
ctgccg 3642236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 422gcggtggcgc tggcggtgcc ggcgctggtg ctgccg
3642336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 423ctggtggtgc
tggcggcggc gccgctggcg gtgccg
3642436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 424ctgattgtgc tggcggcgcc ggcgctggcg
gcgccg 3642536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 425gtgattgtgc tggcggcgcc ggcgctggcg gcgccg
3642636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 426gcggtggtgc
tggcggtgcc ggcgctggcg gtgccg
3642736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 427ctgattattg tggcggcggc gccggcggtg
gcgccg 3642836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 428attgtggcgg tgattgtggc gccggcggtg gcgccg
3642936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 429ctggtggcgc
tggcggcgcc gattattgcg gtgccg
3643036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 430attgcggcgg tgctggcggc gccggcgctg
gtgccg 3643136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 431attgcgctgc tggcggcgcc gattattgcg gtgccg
3643236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 432gcggcgctgg
cgctggtggc gccggtgatt gtgccg
3643336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 433attgcgctgg tggcggcgcc ggtggcgctg
gtgccg 3643436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 434attattgtgg cggtggcgcc ggcggcgatt gtgccg
3643536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 435gcggtggcgg
cgattgtgcc ggtgattgtg gcgccg
3643636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 436gcggtgctgg tgctggtggc gccggcggcg
gcgccg 3643736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 437gtggtggcgc tgctggcgcc gctgattgcg gcgccg
3643836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 438gcggcggtgg
tgattgcgcc gctgctggcg gtgccg
3643936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 439attgcggtgg cggtggcggc gccgctgctg
gtgccg 3644036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 440ctggtggcga ttgtggtgct gccggcggtg gcgccg
3644136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 441gcggtggcga
ttgtggtgct gccggcggtg gcgccg
3644236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 442gcggtgattc tgctggcgcc gctgattgcg
gcgccg 3644336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 443ctggtgattg cgctggcggc gccggtggcg ctgccg
3644436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 444gtgctggcgg
tggtgctgcc ggcggtggcg ctgccg
3644536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 445gtgctggcgg tggcggcgcc ggcggtgctg
ctgccg 3644636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 446gcggcggtgg tgctgctgcc gattattgcg gcgccg
3644736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 447gcgctgctgg
tgattgcgcc ggcgattgcg gtgccg
3644836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 448gcggtgctgg tgattgcggt gccggcgatt
gcgccg 3644936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 449gcgctgctgg tggtgattgc gccgctggcg gcgccg
3645036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 450gtgctggtgg
cggcgattct gccggcggcg attccg
3645136DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 451gtgctggtgg cggcggtgct gccgattgcg
gcgccg 3645236DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 452gtgctggcgg cggcggtgct gccgctggtg gtgccg
3645336DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 453gcgattgcga
ttgtggtgcc ggcggtggcg gtgccg
3645436DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 454gtggcgatta ttgcggtgcc ggcggtggtg
gcgccg 3645536DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 455attgtggcgc tggtggcgcc ggcggcggtg gtgccg
3645636DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 456gcggcgattg
tgctgctgcc ggcggtggtg gtgccg
3645736DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 457gcggcgctga ttgtggtgcc ggcggtggcg
gtgccg 3645836DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 458gcgattgcgc tggtggtgcc ggcggtggcg gtgccg
3645936DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 459ctggcgattg
tgccggcggc gattgcggcg ctgccg
3646036DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 460ctggtggcga ttgcgccggc ggtggcggtg
ctgccg 3646136DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 461gtgctggcgg tggcgccggc ggtggcggtg ctgccg
3646236DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 462attctggcgg
tggtggcgat tccggcggcg gcgccg
3646336DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 463attctggtgg cggcggcgcc gattgcggcg
ctgccg 3646436DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 464attctggcgg tggcggcgat tccggcggcg ctgccg
3646536DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 465gtgattgcga
ttccggcgat tctggcggcg gcgccg
3646636DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 466gcgattatta ttgtggtgcc ggcgattgcg
gcgccg 3646736DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 467gcgattctga ttgtggtggc gccgattgcg gcgccg
3646836DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 468gcggtgattg
tgccggtggc gattattgcg gcgccg
3646936DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 469gcggtggtga ttgcgctgcc ggcggtggtg
gcgccg 3647036DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 470gcgctggtgg cggtgattgc gccggtggtg gcgccg
3647136DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 471gcgctggtgg
cggtgctgcc ggcggtggcg gtgccg
3647236DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 472gcgctggtgg cgccgctgct ggcggtggcg
gtgccg 3647336DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 473gcggtgctgg cggtggtggc gccggtggtg gcgccg
3647436DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 474gcggtgattg
cggtggcgcc gctggtggtg gcgccg
3647536DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 475gcggtgattg cgctggcgcc ggtggtggtg
gcgccg 3647636DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 476gtggcgattg cgctggcgcc ggtggtggtg gcgccg
3647736DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 477gtggcgctgg
cgctggcgcc ggtggtggtg gcgccg
3647836DNAArtificial SequenceThe polynucleotide sequence that encodes
advanced Macromolecule Transduction Domain (aMTD) peptide for
Improvement of Cell-Permeability 478gtggcggcgc tgctgccggc ggtggtggtg
gcgccg 3647936DNAArtificial SequenceThe
polynucleotide sequence that encodes advanced Macromolecule
Transduction Domain (aMTD) peptide for Improvement of
Cell-Permeability 479gtggcgctgg cgctgccggc ggtggtggtg gcgccg
3648036DNAArtificial SequenceThe polynucleotide sequence
that encodes advanced Macromolecule Transduction Domain (aMTD)
peptide for Improvement of Cell-Permeability 480gtggcgctgc
tggcgccggc ggtggtggtg gcgccg 36
User Contributions:
Comment about this patent or add new information about this topic: