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Pharmaceutical Research

, 36:184 | Cite as

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

  • Bastian Quaas
  • Laura Burmeister
  • Zhaopeng Li
  • Alexandra Satalov
  • Peter Behrens
  • Andrea Hoffmann
  • Ursula RinasEmail author
Research Paper
  • 168 Downloads

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.

KEY WORDS

protein aggregation protein solubility protein stability recombinant human bone morphogenetic protein-2 refolding 

ABBREVIATIONS

BRE-Luc

BMP responsive element luciferase

CHO

Chinese hamster ovary

DLS

Dynamic light scattering

E. coli

Escherichia coli

Gdn-HCl

Guanidine hydrochloride

nRFU

normalized relative fluorescence units

nRLU

normalized relative light units

pI

Isoelectric point

rhBMP-2

Recombinant human bone morphogenetic protein 2

RLU

Relative light units

TGF-β

Transforming growth factor β

Tm

Denaturation (melting) temperature

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

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.

Supplementary material

11095_2019_2705_MOESM1_ESM.docx (169 kb)
ESM 1 (DOCX 169 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Bastian Quaas
    • 1
  • Laura Burmeister
    • 2
    • 3
  • Zhaopeng Li
    • 1
  • Alexandra Satalov
    • 4
    • 5
  • Peter Behrens
    • 3
    • 4
  • Andrea Hoffmann
    • 2
    • 3
  • Ursula Rinas
    • 1
    • 6
    Email author
  1. 1.Leibniz University of Hannover, Technical Chemistry – Life ScienceHannoverGermany
  2. 2.Hannover Medical School, Department of Orthopedic Surgery Graded Implants and Regenerative Strategies, Laboratory of Biomechanics and BiomaterialsHannoverGermany
  3. 3.Lower Saxony Center for Biomedical Engineering Implant Research and DevelopmentHannoverGermany
  4. 4.Leibniz University Hannover, Institute for Inorganic ChemistryHannoverGermany
  5. 5.Pasargad Institute for Advanced Innovative SolutionsTehranIran
  6. 6.Helmholtz Centre for Infection ResearchBraunschweigGermany

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