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Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis
Joint Authors
Gul, Roquyya
Hanif, Muhammad Umair
Hanif, Muhammad Imran
Hashmi, Abid Ali
Source
Issue
Vol. 2017, Issue 2017 (31 Dec. 2017), pp.1-11, 11 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2017-11-23
Country of Publication
Egypt
No. of Pages
11
Main Subjects
Abstract EN
Recombinant human Bone Morphogenetic Protein 2 (rhBMP2) has important applications in the spine fusion and ortho/maxillofacial surgeries.
Here we first report the secretory expression of biological active dimerized rhBMP2 from Bacillus subtilis system.
The mature domain of BMP2 gene was amplified from pTz57R/BMP2 plasmid.
By using pHT43 expression vector two constructs, pHT43-BMP2-M (single BMP2 gene) and pHT43-BMP2-D (two BMP2 genes coupled with a linker to produce a dimer), were designed.
After primary cloning (DH5α strain) and sequence analysis, constructs were transformed into Bacillus subtilis for secretory expression.
Expression conditions like media (2xYT) and temperature (30°C) were optimized.
Maximum 35% and 25% secretory expression of monomer (~13 kDa) and dimer (~25 kDa), respectively, were observed on SDS-PAGE in SCK6 strain.
The expression and dimeric nature of rhBMP2 were confirmed by western blot and native PAGE analysis.
For rhBMP2 purification, 200 ml culture supernatant was freeze dried to 10 ml and dialyzed (Tris-Cl, pH 8.5) and Fast Protein Liquid Chromatography (6 ml, Resource Q column) was performed.
The rhBMP2 monomer and dimer were eluted at 0.9 M and 0.6 M NaCl, respectively.
The alkaline phosphatase assay of rhBMP2 (0, 50, 100, 200, and 400 ng/ml) was analyzed on C2C12 cells and maximum 200 ng/ml activity was observed in dose dependent manner.
American Psychological Association (APA)
Hanif, Muhammad Umair& Gul, Roquyya& Hanif, Muhammad Imran& Hashmi, Abid Ali. 2017. Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis. BioMed Research International،Vol. 2017, no. 2017, pp.1-11.
https://search.emarefa.net/detail/BIM-1139485
Modern Language Association (MLA)
Hanif, Muhammad Umair…[et al.]. Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis. BioMed Research International No. 2017 (2017), pp.1-11.
https://search.emarefa.net/detail/BIM-1139485
American Medical Association (AMA)
Hanif, Muhammad Umair& Gul, Roquyya& Hanif, Muhammad Imran& Hashmi, Abid Ali. Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis. BioMed Research International. 2017. Vol. 2017, no. 2017, pp.1-11.
https://search.emarefa.net/detail/BIM-1139485
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-1139485