Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

João E Oliveira,Paolo Bartolini,Luís A V D Pereira,Eliana R Lima,Miriam F Suzuki,Renata Damiani,Geraldo S Magalhães,Leonardo P Faverani,Kleicy C Amaral,Anderson M S Santos

doi:10.3390/cells10123525

Abstract

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Highlights

It is important to note that the presence of the temperature-sensitive repressor should potentially stop Human bone morphogenetic protein-2 (hBMP-2) synthesis at 30 ◦ C, favoring bacterial growth, allowing its maximum expression at 42 ◦ C, with
It is important to note that the presence of the temperature-sensitive repressor should potentially stop hBMP-2 synthesis at 30 °C, favoring bacterial growth, allowing its maximum expression at 42 °C, with repressor deactivation
An original expression vector based on hBMP-2 cDNA, the λPL promoter, the DsbA signal sequence, and the ampicillin resistance (AmpR)

Summary

Introduction

Human bone morphogenetic protein-2 (hBMP-2) was first discovered when Urist et al. Established the biological basis of morphogenesis [1,2,3]. Its purification from demineralized bone matrix and definitive characterization were first performed by Reddi et al [4,5,6]. HBMP-2 is a homodimeric cysteine-knot protein that belongs to the transforming growth factor-β (TGF-β) family, whose structure is stabilized through dimerization and an additional intermolecular disulfide bond [7,8]. It was soon observed that hBMP-2 is one of the most efficient osteoinductors ever described, especially because of its capacity of inducing bone regeneration and ectopic bone formation in adult vertebrates.

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