Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.
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.
Vallejo
Luis Felipe
LF
Biochemical Engineering Division, National Research Center for Biotechnology (GBF), Mascheroder Weg 1, 38124, Braunschweig, Germany.
Brokelmann
Maren
M
Marten
Sabine
S
Trappe
Susanne
S
Cabrera-Crespo
Joaquin
J
Hoffmann
Andrea
A
Gross
Gerhard
G
Weich
Herbert A
HA
Rinas
Ursula
U
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
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta