title: Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.
abstract: Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.
authors.affiliation: Biochemical Engineering Division, National Research Center for Biotechnology (GBF), Mascheroder Weg 1, 38124, Braunschweig, Germany.
authors.forename: Luis Felipe
authors.initials: LF
authors.lastname: Vallejo
authors.forename: Maren
authors.initials: M
authors.lastname: Brokelmann
authors.forename: Sabine
authors.initials: S
authors.lastname: Marten
authors.forename: Susanne
authors.initials: S
authors.lastname: Trappe
authors.forename: Joaquin
authors.initials: J
authors.lastname: Cabrera-Crespo
authors.forename: Andrea
authors.initials: A
authors.lastname: Hoffmann
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gross
authors.forename: Herbert A
authors.initials: HA
authors.lastname: Weich
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
mesh_terms: D019149:Bioreactors; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005818:Genetic Engineering; D002479:Inclusion Bodies / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016212:Transforming Growth Factor beta
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta