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Keywords:

  • human bone morphogenetic protein;
  • bacterial expression;
  • heparin binding;
  • limb bud;
  • proteoglycan synthesis;
  • variant bone morphogenetic protein 2

Abstract

  1. Top of page
  2. Abstract
  3. References

Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP-2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP-2 protein of Mr 26000 was prepared with a purity greater 98%. The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15–20 nM). A peptide 1–17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N-terminal reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1–12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1–17. A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible. These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity.

Abbreviations
BMP

bone morphogenetic protein

EC50

effector concentration resulting in half-maximal response

Rmax

maximal response obtained at saturation levels

TGF

transforming growth factor

CHO

Chinese hamster ovary

FGF

fibroblast-growth factor

References

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