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

Autor: Bastian Quaas, Laura Burmeister, Zhaopeng Li, Andrea Hoffmann, Peter Behrens, Ursula Rinas, Alexandra Satalov
Přispěvatelé: HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Rok vydání: 2019
Předmět:
recombinant human bone morphogenetic protein-2
Protein Folding
Protein Conformation
Bone Morphogenetic Protein 2
Pharmaceutical Science
02 engineering and technology
Sodium Chloride
Protein aggregation
030226 pharmacology & pharmacy
Bone morphogenetic protein 2
Inclusion bodies
protein aggregation
refolding
Divalent
Protein Aggregates
03 medical and health sciences
0302 clinical medicine
Transforming Growth Factor beta
Escherichia coli
Humans
Pharmacology (medical)
Thermal stability
Particle Size
Solubility
Pharmacology
chemistry.chemical_classification
Oxalates
Heparin
Protein Stability
Osmolar Concentration
Organic Chemistry
Temperature
Biological activity
protein solubility
Hydrogen-Ion Concentration
021001 nanoscience & nanotechnology
Recombinant Proteins
Chaotropic agent
protein stability
chemistry
Biophysics
Molecular Medicine
0210 nano-technology
Biotechnology
Zdroj: Pharmaceutical research
ISSN: 1573-904X
0724-8741
DOI: 10.1007/s11095-019-2705-5
Popis: 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.
Databáze: OpenAIRE
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