Bastian Quaas1, Laura Burmeister2,3, Zhaopeng Li1, Alexandra Satalov4,5, Peter Behrens3,4, Andrea Hoffmann2,3, Ursula Rinas6,7. 1. Leibniz University of Hannover, Technical Chemistry - Life Science, Hannover, Germany. 2. Hannover Medical School, Department of Orthopedic Surgery Graded Implants and Regenerative Strategies, Laboratory of Biomechanics and Biomaterials, Hannover, Germany. 3. Lower Saxony Center for Biomedical Engineering Implant Research and Development, Hannover, Germany. 4. Leibniz University Hannover, Institute for Inorganic Chemistry, Hannover, Germany. 5. Pasargad Institute for Advanced Innovative Solutions, Tehran, Iran. 6. Leibniz University of Hannover, Technical Chemistry - Life Science, Hannover, Germany. Ursula.Rinas@helmholtz-hzi.de. 7. Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124, Braunschweig, Germany. Ursula.Rinas@helmholtz-hzi.de.
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.
PURPOSE: There is a plethora of studies on recombinant humanbone 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.
Entities:
Keywords:
protein aggregation; protein solubility; protein stability; recombinant human bone morphogenetic protein-2; refolding
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