Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells.

Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor-ß (TGF-ß) super family of proteins and is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post-translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half-life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in-vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in-vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO-K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production.

Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design.

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Fabricating a novel HLC-hBMP2 fusion protein for the treatment of bone defects.

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Effects of human bone morphogenetic protein 2 (hBMP2) on tertiary dentin formation.

Formation of tertiary dentin to maintain pulp vitality is a major odontoblastic response to dental pulp injury. Human bone morphogenetic protein 2 (hBMP2) can promote proliferation and differentiation of odontoblasts. Current study is interested in evaluating if the hBMP2 can promote the regeneration of tertiary dentin and cure dental pulp injury using the adenoviral vector to deliver hBMP2 cDNA into the pulp. Primary culture of dental pulp cells of exfoliated deciduous teeth (hDPCs) was established. Human serotype 5 adenoviral vector, AdCMV-hBMP2, was created. AdCMV-hBMP2 was used to transduce hDPCs in vitro and dental pulp cells in animal model in vivo. Data clearly demonstrated that hBMP2 increased ALP and mineralization. Reverse transcription-real time quantitative PCR (RT-QPCR) data showed that hBMP2 dramatically increased gene expressions of Runx2 (Runt-related transcription factor 2), ALP, Col Iα (Collagen 1a1), SP7 (Osterix), DMP1 (dentin matrix acidic phosphoprotein 1), DSPP (dentin sialophosphoprotein), and BSP (bonesialoprotein), which are normally involved in osteogenesis/odontogenesis. Data from in vivo assays demonstrated that hBMP2 promoted pulp cell proliferation and increased formation of tertiary dentin in dental pulp. Our in vitro and in vivo data suggest that hBMP2 gene can efficiently be delivered into the dental pulp cells by adenovirus, and show potential clinical application for the treatment of dental pulp damage.

Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli.

Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.

Novel Synthesized Nanofibrous Scaffold Efficiently Delivered hBMP-2 Encoded in Adenoviral Vector to Promote Bone Regeneration.

Treatment of bone defect, especially large bone defect, is still a challenge for physicians clinically. Bone morphogenetic protein 2 (BMP-2) can induce osteoblast differentiation and promote new bone formation. Recently, nanomaterials have been widely used as a carrier to hold and deliver biomolecules, like human bone morphogenetic protein 2 gene (hBMP-2) in target cells/tissues. Most nanomethods, however, need further modification in order to work more reliably in clinical applications. Therefore, in this study, we created a novel poly(lactic-co-glycolic acid [PLGA]) nanofibrous scaffold using an electrospinning technique; then, using a lyophilization process to allow nanofibrous scaffold to adsorb hBMP-2 adenoviral vector, AdCMV-hBMP2. Results indicate that the lyophilized poly(lactic-co-glycolic acid) nanofibrous scaffold/AdCMVhBMP2 can efficiently release and transduce cells in vitro and in vivo, and secrete functional hBMP-2 to promote osteogenic differentiation in vitro, and new bone generation in vivo. Importantly, the amount of newly formed bone covered >80% of the bone defect area 8 weeks post-implantation in vivo, in which the defect could not be repaired without any treatment in general. Our data demonstrate that the lyophilized PLGA nanofibrous scaffold/AdCMV-hBMP2 created herein stably and efficiently release functional viral vector to transduce local cells, resulting in secretion of hBMP-2 and promote new bone formation in vivo. Our new nanodelivery method has potential clinical application for the repair of large bone defects.

Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor.

The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40 µg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing.

Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

Rehabilitation effect of BMSCs that was transfected with hBMP-2 through adenovirus combination with DBM on rabbit osteonecrosis in femoral head / 天津医药

Objective To evaluate the effect of human bone morphogenetic protein 2 (hBMP-2)/Bone Mesenchymal Stem Cells (BMSCs)/demineralized bone matrix(DBM) on repairing rabbits’femoral head after necrosis and to explore the new treatments for femoral head necrosis. Methods Femoral head necrosis models was established by clinical core decom?pression combined with liquid nitrogen frozen. Then, animals were randomly devided into 4 groups (n=12 per group):Group A were not implanted anything as control group, Group B were implanted with DBM. Group C were implanted with hBMP-2/DBM. Group D were implanted with hBMP-2/BMSCs/DBM. Four rabbits from each group were sacrificed at 4,8 and 12 weeks after surgery to evaluate the the repairing effect of Osteonecrosis of the femoral head (ONFH) through X-ray examina?tion, observation of the specimen and HE staining. Results X-ray revealed defect of femoral head in Group A without clear bone formation. There is a little fibrous hyperplasia and no obvious osteogenic response. By contrast, the femoral head defect areas became fuzzy in group B, group C and group D with new bone trabeculars. And the regenerate phenomenons of group D were significantly better than that of group B and group C of the same time point. As to the Lane-Sandhu X Ray scores, it is lower in group A than that in group B;It is lower in group C than that in group D(P<0.05). There is no statistical difference between Group B and Group C. General observation of the specimen revealed that the femoral head of group A collapsed with drilling holes. The femoral heads of group B and group C showed no collapse but the drilling holes existed. Femoral head in group D was not collapsed and the drilling holes disappeared. HE staining showed that bone trabeculars became ne?crotic and fragmented in Group A with a lot of air trapped cells. There were newborn immature bone trabeculars and osteo? blasts in group B and group C. Group D were of large number of bone cells, fat cells, and newborn mature bone trabeculars. The ratio of empty lacuna is higher in Group A than that in Group B;it is higher in Group C than that in Group D(P<0.05). Conclusion hBMP-2/BMSCs/DBM can induce BMSCs differentiation into osteoblasts after being implanted. It has good re?pairing effect on ONFH with good application prospect.

Expression and mechanism of cDNA of human bone morphogenetic protein 2 via eukaryotic expression vector in rabbit mesenchymal stem cells / 中华创伤骨科杂志

0.05). Conclusion The recombinant plasmid containing hBMP- 2 cDNA can be transfected and expressed effectively in rabbit MSCs