CROSS-REFERENCE TO RELATED APPLICATIONS 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 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 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).
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).
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).
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.
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.
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 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
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.
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 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.
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.
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.
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.
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.
The combinational treatment of CP-BMP2 and CP-BMP7 synergistically enhance in vitro osteogenic differentiation and in vivo bone regeneration.
The CP-BMP2 and CP-BMP7 can be applied to bone injured area by simple injection without additional vehicles or scaffolds.
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 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.
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.
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)
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.
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).
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
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.
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).
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).
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
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.
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).
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).
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.
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.
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).
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.
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.
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).
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.
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).
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.
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).
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.
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.
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).
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.
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.
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).
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.
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).
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.
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.
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.
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.
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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.
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.
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
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.
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).
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.
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 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.
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 2-1. Novel Hydrophobic Cell-Penetrating Peptide (CPP)—Advanced Macromolecule Transduction Domain (aMTD)
2-1-1. Analysis of Hydrophobic CPPs 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.
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.
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 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) 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) 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) 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) 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) 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’ 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.
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.
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.
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).
1. Amino Acid Length: 9-13
2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence
3. Rigidity/Flexibility (Instability Index: II): 40-60
4. Structural Feature (Aliphatic Index: AI): 180-220
5. Hydropathy (GRAVY): 2.1-2.6
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
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.
1. Amino Acid Length: 12
2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence
3. Rigidity/Flexibility (Instability Index: II): 41.3-57.3
4. Structural Feature (Aliphatic Index: AI): 187.5-220.0
5. Hydropathy (GRAVY): 2.2-2.6
6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids—A, V, L, I and P
TABLE 2-1
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 1-46)]
TABLE 2-2
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 47-92)]
TABLE 2-3
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 93-138)]
TABLE 2-4
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 139-184)]
TABLE 2-5
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 185-230)]
TABLE 2-6
[Newly Developed Hydrophobic CPPs-240 aMTDs that all Critical Factors are
Considered and Satisfied (aMTD 231-240)]
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 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
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 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
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
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
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).
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
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).
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 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 3-1. aMTD/SD-Fused CP-BMP2/7 Show Great Cell-Permeability.
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.
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,
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.
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. 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. 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.
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. 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).
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. 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. 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.
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. 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 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 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 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 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 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 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.
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 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 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 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 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 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 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 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 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.
7-2. Differentiation of Cells
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.
7-3. Phosphorylation of Smad Signaling
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.
7-4. Measurement of Alkaline Phosphatase Activity
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.
7-5. Measurement of Calcium Content
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.
7-6. Alizarin Red S Staining
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) 8-1. In Vivo Calvarial Critical Sized Defect Model
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.
8-2. Calvarial Injection Assay
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.
8-3. Soft X-Ray
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.
8-4. 3D micro-CT
Three-dimensional images from micro-CT scanning were analyzed with Adobe Photoshop CS6 (Adobe Systems, CA, USA) to measure regenerated bone areas.
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).
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.
[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
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