Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2

Thermoresponsive coatings of poly(N-isopropylacrylamide-co-DMAEMA)/cellulose sulfate (PNIPAM-DMAEMA/CS) complexes are reported eluting bone-morphogenetic-protein-2 (BMP-2) on demand relevant for implant assisted local bone healing. PNIPAM-DMAEMA/CS dispersions contained colloid particles with hydrodynamic radii RH = 170–288 nm at T = 25 °C shrinking to RH = 74–103 nm at T = 60 °C. Obviously, PNIPAM-DMAEMA/CS undergoes volume phase transition (VPT) analogously to pure PNIPAM, when critical VPT temperature (VPTT) is exceeded. Temperature dependent turbidity measurements revealed broad VPT and VPTT 47 °C for PNIPAM-DMAEMA/CS colloid dispersions at pH = 7.0. FTIR spectroscopy on thermoresponsive PNIPAM-DMAEMA/CS particle coatings at germanium model substrates under HEPES buffer indicated both wet-adhesiveness and VPT behavior based on diagnostic band intensity increases with temperature. From respective temperature courses empirical VPTT ≈ 42 °C for PNIPAM-DMAEMA/CS coatings at pH = 7.0 were found, which were comparable to VPTT found for respective dispersions. Finally, the PNIPAM-DMAEMA/CS coatings were loaded with BMP-2 and model protein papain (PAP). Time dependent FTIR spectroscopic measurements showed, that for T = 37 °C there was a relative protein release of ≈30% for PAP and ≈10% for BMP-2 after 24 h, which did not increase further. Heating to T = 42 °C for PAP and to 47 °C for BMP-2 further secondary protein release of ≈20% after 24 h was found, respectively, interesting for clinical applications. BMP-2 eluted even at 47 °C was found to be still biologically active.

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P121 EXPRESSION OF HUMAN BONE MORPHOGENETIC PROTEIN-2 AS A BIOLOGICALLY ACTIVE SOLUBLE PROTEIN IN ESCHERICHIA COLI

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(07)60573-1 ◽

2006 ◽

Vol 14 ◽

pp. S76

Author(s):

J.-W. Huh ◽

H. Ihm ◽

M.M. Choi ◽

S.W. Cho

Keyword(s):

Escherichia Coli ◽

Bone Morphogenetic Protein ◽

Soluble Protein ◽

Human Bone ◽

Biologically Active ◽

Bone Morphogenetic Protein 2 ◽

Morphogenetic Protein ◽

Human Bone Morphogenetic Protein

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Bone Morphogenetic Protein-2 Conjugated to Quantum Dot®s is Biologically Functional

Nanomaterials ◽

10.3390/nano10061208 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1208

Author(s):

Daniel Halloran ◽

Vrathasha Vrathasha ◽

Hilary W. Durbano ◽

Anja Nohe

Keyword(s):

Quantum Dot ◽

Bone Morphogenetic Protein ◽

Fluorescent Dyes ◽

Fluorescein Isothiocyanate ◽

Biologically Active ◽

Bone Morphogenetic Protein 2 ◽

Bmp Receptors ◽

Morphogenetic Protein ◽

Long Time ◽

Neural Growth

Quantum Dot®s (QDot®s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot®s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot®s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot®s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot®s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog’s stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.

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Potentiation of Astrogliogenesis by STAT3-Mediated Activation of Bone Morphogenetic Protein-Smad Signaling in Neural Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.02435-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4931-4937 ◽

Cited By ~ 82

Author(s):

Shinji Fukuda ◽

Masahiko Abematsu ◽

Hiroyuki Mori ◽

Makoto Yanagisawa ◽

Tetsushi Kagawa ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Bone Morphogenetic Protein ◽

Negative Regulator ◽

Bone Morphogenetic Protein 2 ◽

Cytokine Signaling ◽

Morphogenetic Protein ◽

Regulatory Loop ◽

Bmp Antagonist

ABSTRACT Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.

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