scholarly journals 1-Step Versus 2-Step Immobilization of Alkaline Phosphatase and Bone Morphogenetic Protein-2 onto Implant Surfaces Using Polydopamine

2013 ◽  
Vol 19 (8) ◽  
pp. 610-619 ◽  
Author(s):  
Arnold W.G. Nijhuis ◽  
Jeroen J.J.P. van den Beucken ◽  
Otto C. Boerman ◽  
John A. Jansen ◽  
Sander C.G. Leeuwenburgh
Keyword(s):  
Alkaline Phosphatase ◽  
Bone Morphogenetic Protein ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein

Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli

2019 ◽  
Vol 32 (3) ◽  
pp. 153-157
Author(s):  
Wei Chen ◽  
Caiqian Zhang ◽  
Yeqing Wu ◽  
Xiuping Su
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Bone Morphogenetic Protein ◽  
Human Bone ◽  
Bone Morphogenetic Protein 2 ◽  
E Coli ◽  
Alp Activity ◽  
Morphogenetic Protein ◽  
Human Bone Morphogenetic Protein ◽  
Recombinant Human Bone

Abstract We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2 mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500 ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.

scholarly journals Nephroblastoma Overexpressed (Nov) Inactivation Sensitizes Osteoblasts to Bone Morphogenetic Protein-2, But Nov Is Dispensable for Skeletal Homeostasis

Endocrinology ◽  
2010 ◽  
Vol 151 (1) ◽  
pp. 221-233 ◽  
Author(s):  
Ernesto Canalis ◽  
Anna Smerdel-Ramoya ◽  
Deena Durant ◽  
Aris N. Economides ◽  
Wesley G. Beamer ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Bone Morphogenetic Protein ◽  
Tissue Growth ◽  
Bone Morphogenetic Protein 2 ◽  
Wild Type ◽  
Reporter Construct ◽  
Mineral Density ◽  
Morphogenetic Protein ◽  
Skeletal Homeostasis ◽  
Trabecular Number

Abstract Overexpression of nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov family of proteins, inhibits osteoblastogenesis and causes osteopenia. The consequences of Nov inactivation on osteoblastogenesis and the postnatal skeleton are not known. To study the function of Nov, we inactivated Nov by homologous recombination. Nov null mice were maintained in a C57BL/6 genetic background after the removal of the neomycin selection cassette and compared with wild-type controls of identical genetic composition. Nov null mice were identified by genotyping and absent Nov mRNA in calvarial extracts and osteoblast cultures. Nov null mice did not exhibit developmental skeletal abnormalities or postnatal changes in weight, femoral length, body fat, or bone mineral density and appeared normal. Bone volume and trabecular number were decreased only in 1-month-old female mice. In older mice, after 7 months of age, osteoblast surface and bone formation were increased in females, and osteoclast and eroded surfaces were increased in male Nov null mice. Calvarial osteoblasts from Nov null mice displayed enhanced alkaline phosphatase activity, alkaline phosphatase mRNA, and transactivation of a bone morphogenetic protein (BMP)/phosphorylated mothers against decapentaplegic reporter construct in response to BMP-2. Similar results were obtained after the down-regulation of Nov by RNA interference in ST-2 stromal and MC3T3 cells. Osteoclast number was increased in marrow stromal cell cultures from Nov null mice. Surface plasmon resonance demonstrated direct interactions between Nov and BMP-2. In conclusion, Nov sensitizes osteoblasts to BMP-2, but Nov is dispensable for the maintenance of bone mass.

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