Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2

authored by: Bastian Quaas, Laura Burmeister, Zhaopeng Li, Alexandra Satalov, Peter Behrens, Andrea Hoffmann, Ursula Rinas
Abstract: Purpose: 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.
Organisation(s): Institute of Technical Chemistry
Institute of Inorganic Chemistry
External Organisation(s): Hannover Medical School (MHH)
Helmholtz Centre for Infection Research (HZI)
NIFE - Lower Saxony Centre for Biomedical Engineering, Implant Research and Development
Type: Article
Journal: Pharmaceutical research
Volume: 36
ISSN: 0724-8741
Publication date: 20.11.2019
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Biotechnology, Molecular Medicine, Pharmacology, Pharmaceutical Science, Organic Chemistry, Pharmacology (medical)
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Electronic version(s): http://hdl.handle.net/10033/622106 (Access: Open)
https://doi.org/10.1007/s11095-019-2705-5 (Access: Closed)