Expression and characterization of a human BMP-7 variant with improved biochemical properties

Swencki-Underwood, Bethany; Mills, Juliane K.; Vennarini, Joe; Boakye, Ken; Luo, Jinquan; Pomerantz, Steven C.; Cunningham, Mark R.; Farrell, Francis X.; Naso, Michael F.; Amegadzie, Bernard Y.

doi:10.1016/j.pep.2007.09.016

Cited by 36 publications

(20 citation statements)

References 18 publications

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“…37 In a manner similar to that described previously, 37 we introduced mutations into the N terminus of the BMP7 prodomain to enhance prodomain and mature domain cleavage. In addition, a random mutagenesis approach was used that introduced point mutations onto the surface of the mature domain.…”

Section: Discussionmentioning

confidence: 99%

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

et al. 2012

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Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-b superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.

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Section: Discussionmentioning

confidence: 99%

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

et al. 2012

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“…However, like many TGF-β proteins, BMP-7 is poorly made and processed in mammalian cells (Swencki-Underwood et al, 2008), and upon delivery, is likely rapidly cleared from the blood (Coffey et al, 1987). To address these limitations, Sugimoto et al (2012), developed a small molecule BMP-7 mimetic (AA123), which has the same anti-fibrotic activity as recombinant BMP-7 in a mouse model of kidney disease.…”

Section: Targeting Tgf-β Signaling For the Treatment Of Fibrosismentioning

confidence: 99%

Targeting TGF-β Mediated SMAD Signaling for the Prevention of Fibrosis

2017

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Fibrosis occurs when there is an imbalance in extracellular matrix (ECM) deposition and degradation. Excessive ECM deposition results in scarring and thickening of the affected tissue, and interferes with tissue and organ homeostasis – mimicking an exaggerated “wound healing” response. Many transforming growth factor-β (TGF-β) ligands are potent drivers of ECM deposition, and additionally, have a natural affinity for the ECM, creating a concentrated pool of pro-fibrotic factors at the site of injury. Consequently, TGF-β ligands are upregulated in many human fibrotic conditions and, as such, are attractive targets for fibrosis therapy. Here, we will discuss the contribution of TGF-β proteins in the pathogenesis of fibrosis, and promising anti-fibrotic approaches that target TGF-β ligands.

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“…Based on the observation that rhBMP-7 displays high affinity for heparin [17], we purified this protein in a single step, using affinity chromatography, with considerable amounts of the dimeric form being obtained without any additional refolding and assembly steps. Since the protein has four potential predicted N-linked glycosylation sites [27], we investigated the possible glycosylation of the expressed protein using an assay based on N-glycosidase F. We were able to observe a significant difference in the molecular mass when the protein was treated with this exoglycosidase by SDS-PAGE, as revealed by western blotting (Fig. 5).…”

Section: Discussionmentioning

confidence: 97%

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

et al. 2010

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Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

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Expression and characterization of a human BMP-7 variant with improved biochemical properties

Cited by 36 publications

References 18 publications

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

Targeting TGF-β Mediated SMAD Signaling for the Prevention of Fibrosis

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

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