TY - JOUR

T1 - Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2

AU - Quaas, Bastian

AU - Burmeister, Laura

AU - Li, Zhaopeng

AU - Satalov, Alexandra

AU - Behrens, Peter

AU - Hoffmann, Andrea

AU - Rinas, Ursula

N1 - Funding information: The authors gratefully acknowledge funding through the Forschergruppe “Gradierte Implantate” FOR2180 and the Exzellenzcluster “Rebirth” EXC62, both Deutsche Forschungsgemeinschaft (DFG), and excellent technical assistance by Anika Hamm (bioactivity measurements), Graded Implants and Regenerative Strategies. We also want to thank the reviewers for their careful and critical reading which helped a lot to improve the manuscript.

PY - 2019/11/20

Y1 - 2019/11/20

N2 - 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.

AB - 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.

KW - protein aggregation

KW - protein solubility

KW - protein stability

KW - recombinant human bone morphogenetic protein-2

KW - refolding

UR - http://www.scopus.com/inward/record.url?scp=85075398646&partnerID=8YFLogxK

U2 - 10.1007/s11095-019-2705-5

DO - 10.1007/s11095-019-2705-5

M3 - Article

C2 - 31748894

AN - SCOPUS:85075398646

VL - 36

JO - Pharmaceutical research

JF - Pharmaceutical research

SN - 0724-8741

IS - 12

M1 - 184

ER -