Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2.
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AbstractPURPOSE: There is a plethora of studies on recombinant human bone morphogenetic protein-2 (rhBMP-2) application and delivery systems, but surprisingly few reports address the biophysical properties of the protein which are of crucial importance to develop effective delivery systems or to solve general problems related to rhBMP-2 production, purification, analysis and application. METHODS:The solubility, stability and bioactivity of rhBMP-2 obtained by renaturation of E. coli derived inclusion bodies was assessed at different pH and in different buffer systems using (dynamic) light scattering and thermal shift assays as well as intrinsic fluorescence measurements and luciferase based bioassays. RESULTS: rhBMP-2 is poorly soluble at physiological pH and higher. The presence of divalent anions further decreases the solubility even under acidic conditions. Thermal stability analyses revealed that rhBMP-2 precipitates are more stable compared to the soluble protein. Moreover, correctly folded rhBMP-2 is also bioactive as precipitated protein and precipitates readily dissolve under appropriate buffer conditions. Once properly formed rhBMP-2 also retains biological activity after temporary exposure to high concentrations of chaotropic denaturants. However, care should be taken to discriminate bioactive rhBMP-2 precipitates from misfolded rhBMP-2 aggregates, e.g. resolvability in MES buffer (pH 5) and a discrete peak in thermoshift experiments are mandatory for correctly folded rhBMP-2. CONCLUSIONS: Our analysis revealed that E. coli derived rhBMP-2 precipitates are not only bioactive but are also more stable compared to the soluble dimeric molecules. Knowledge about these unusual properties will be helpful to design improved delivery systems requiring lower amounts of rhBMP-2 in clinical applications.
CitationPharm Res. 2019 Nov 20;36(12):184. doi: 10.1007/s11095-019-2705-5.
AffiliationHZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
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- Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration.
- Authors: Vallejo LF, Rinas U
- Issue date: 2004 Mar 20
- Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).
- Authors: Luca L, Capelle MA, Machaidze G, Arvinte T, Jordan O, Gurny R
- Issue date: 2010 May 31
- Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli.
- Authors: Chen W, Zhang C, Wu Y, Su X
- Issue date: 2019 Dec 13
- Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.
- Authors: Vallejo LF, Brokelmann M, Marten S, Trappe S, Cabrera-Crespo J, Hoffmann A, Gross G, Weich HA, Rinas U
- Issue date: 2002 Mar 28
- Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.
- Authors: Ding S, Zhang J, Tian Y, Huang B, Yuan Y, Liu C
- Issue date: 2016 Sep 1
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