Patent Application: US-201514838318-A

Abstract:
the present invention is about direct protein delivery system for bone morphogenetic proteins to enhance bone regeneration without the need of additional delivery carrier which requires open surgery for their implantation . the cell - permeable bmp recombinant proteins are fused with advanced macromolecule transduction domain to give them ability for cell penetration and with solubilization domain to increase their solubility and manufacturing yield . because existing bmp recombinant proteins have short half - life , they have been delivered with polymeric or inorganic vehicles for their sustained release . however , cp - bmps fused to combination of amtd and sd can easily and rapidly penetrate into the cytosol after treating on cells that likes hiding in a shelter from wash off in body fluid , and avoid rapid degradation . for the development of cp - bmp , bmp2 and bmp7 have been selected among various types of bmp family due to their potent osteo - inductivity . both of proteins are produced in mature form as an active domain and pro - peptide form + mp ) for prolonged stability of proteins . in the present art , three strategic steps are used to prove the validity of using cp - bmps on bone regeneration and new bone formation . first , randomly selected amtds and various types of sds have been fused to bmp proteins for determine the best structural composition for highest solubility / yield and cell -/ tissue - permeability . next , amtds are fused to bmp2 and bmp7 proteins with optimized structure to determine the best construct for maximized cell - and tissue - permeability . finally , biological activity of bmp2 , bmp7 and combination of bmp2 and bmp7 recombinant proteins have been evaluated to enhance in vitro osteogenic differentiation and in vivo bone regeneration . the cp - bmps can be applied to repair skeletal injuries which are by bone fracture , osteogenesis imperfecta , and bone extraction .

Description:
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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 ). 2 . bending potential ( proline position : pp ): proline presences in the middle ( from 5 ′ to 8 ′ amino acid ) and at the end of sequence 6 . amino acid composition : hydrophobic and aliphatic amino acids — a , v , l , i and p 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 & amp ; 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 . 2 . bending potential ( proline position : pp ): proline presences in the middle ( from 5 ′ to 8 ′ amino acid ) and at the end of sequence 6 . amino acid composition : hydrophobic and aliphatic amino acids — a , v , l , i and p 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 . 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 fig2 . 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 fig3 , 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 . 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 ) ( fig4 ). 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 ( fig5 ) and cloned for protein expression and purification . fig6 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 ) ( fig7 ). 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 ) ( fig8 ). 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 ( fig9 ) and cloned for protein expression and purification . fig1 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 ) ( fig1 ). 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 ( fig1 ). the expression vectors for each bmp2 lp proteins were successfully constructed and cloned for protein expression and purification ( fig1 ). 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 ( fig1 ). 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 fig1 . 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 ( fig1 ). 2l - 5 and 2l - 5c proteins were successfully expressed and purified with significantly improved solubility ( fig1 ). the relative yield also significantly increased in 2l - 5 and 2l - 5c protein ( 48 and 90 folds , respectively ) compared to 2l - 1 protein ( fig1 ). 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 ( fig1 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 ( fig2 ). 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 ( fig2 ). 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 ( fig2 ). 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 ( fig2 ). 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 fig2 . the expression vectors for newly designed bmp7 lp proteins were successfully constructed and cloned for protein expression and purification ( fig2 ). the 7l - 5 and 7l - 5c proteins were successfully expressed and purified with significantly improved solubility ( fig2 ). the 7l - 5 and 7l - 5c protein showed significant increase of yields ( 78 - and 68 - folds , respectively ) relative to 7l - 1 protein ( fig2 ). 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 ( fig2 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 ( fig3 ). 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 ( fig3 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 ( fig3 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 . 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 ( fig3 ). 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 - 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 ( fig3 ). c2c12 myoblasts are known to differentiate into myotubes under the starvation condition (& lt ; 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 ( fig3 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 fig3 . 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 ( fig4 ). in alp assay , it showed superior alp activity following the combinational treatment of 2m - 4 and 7m - 4 compared to others ( fig4 ). 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 ( fig4 ). 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 ( fig4 ). 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 ( fig4 ). 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 ) ( fig4 ). 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 . 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 ( fig4 ). 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 ( fig4 ). 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 & amp ; e staining . as shown in fig4 , 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 ( fig4 ). 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 ( fig5 ). 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 . 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 . 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 ). 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 . 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 ni 2 + - 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 . 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 . recombinant proteins were conjugated to fluorescein isothiocynate ( fitc ), according to the manufacturer &# 39 ; 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 ). 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 . 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 × 10 5 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 . 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 . 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 . alp activity was measured with cell lysate , according to the manufacturer &# 39 ; 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 . 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 &# 39 ; 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 . mc3t3 - e1 cells and c3h10t1 / 2 cells were plated at 5 × 10 4 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 . 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 . 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 &# 39 ; s trichrome as described in ‘ 4 . 5 . 5 histological analysis ’ section . 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 . 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 &# 39 ; 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 - 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42 ; discussion 42 .