Details
| Original language | English |
|---|---|
| Pages (from-to) | 185-194 |
| Number of pages | 10 |
| Journal | Journal of biotechnology |
| Volume | 94 |
| Issue number | 2 |
| Early online date | 26 Nov 2001 |
| Publication status | Published - 28 Mar 2002 |
| Externally published | Yes |
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.
Keywords
- E. coli, In vitro refolding, Purification, rhBMP-2
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Journal of biotechnology, Vol. 94, No. 2, 28.03.2002, p. 185-194.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli
AU - Vallejo, Luis Felipe
AU - Brokelmann, Maren
AU - Marten, Sabine
AU - Trappe, Susanne
AU - Cabrera-Crespo, Joaquin
AU - Hoffmann, Andrea
AU - Gross, Gerhard
AU - Weich, Herbert A.
AU - Rinas, Ursula
N1 - Funding Information: L.F. Vallejo acknowledges a scholarship from COLCIENCIAS (Colombian Institute for Science and Technology). J. Cabrera-Crespo thanks FAPESP (The State S. Paulo Research Foundation, No. 99/05554-5) and the Foundation Institute Butantan (S. Paulo, Brasil) for a post-doctoral scholarship. This work was supported in part by the SFB 578 and DFG grants We 1211/4-1. We are also thankful to J. van den Heuvel for advice concerning primer selection and expression vector construction.
PY - 2002/3/28
Y1 - 2002/3/28
N2 - 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.
AB - 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.
KW - E. coli
KW - In vitro refolding
KW - Purification
KW - rhBMP-2
UR - http://www.scopus.com/inward/record.url?scp=0037187447&partnerID=8YFLogxK
U2 - 10.1016/S0168-1656(01)00425-4
DO - 10.1016/S0168-1656(01)00425-4
M3 - Article
C2 - 11796171
AN - SCOPUS:0037187447
VL - 94
SP - 185
EP - 194
JO - Journal of biotechnology
JF - Journal of biotechnology
SN - 0168-1656
IS - 2
ER -