Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding

Abstract

Recombinant human bone morphogenetic protein-2 (rhBMP-2), a cystine-knot containing disulfide linked homodimer, was produced in form of inclusion bodies (IBs) using E. coli BL21 (DE3) and SHuffle T7 Express. Non-reducing SDS-PAGE analysis revealed that rhBMP-2 was present within IBs in both strains as monomer and in form of disulfide-linked dimers. Purified dimeric disulfide-linked rhBMP-2 was obtained from IBs by two different methods. The first method involved classical solubilisation using strong denaturants and subsequent refolding. The second method involved mild extraction without refolding. Both rhBMP-2 dimer variants were purified by Heparin-affinity chromatography. Mildly extracted rhBMP-2 was further purified by size-exclusion chromatography. The resulting dimeric rhBMP-2 variants were studied regarding bioactivity and folding status. In contrast to the rhBMP-2 dimer obtained by classical refolding it was shown that the disulfide-linked dimer obtained by mild extraction was not correctly folded e.g. the hydrophobic core not correctly formed. Moreover, refolded dimeric rhBMP-2 was bioactive and the mildly extracted dimeric rhBMP-2 did not show any bioactivity. Disulfide-bond analysis revealed that the intricate disulfide-bond pattern of the complex cystine-knot scaffold was not present in the IB embedded disulfide-linked rhBMP-2 dimer but was formed later during classical refolding of the reduced protein under appropriate redox conditions.

Varying the sustained release of BMP‐2 from chitosan nanogel‐functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications

“…3.5), the assay was performed in a 96 well‐plate format as described in (Quaas et al, 2019) and for the quantification of BMP‐2 activity after release (cf. 3.4) in a 24 well‐plate format as described in (Quaas et al, 2018).…”

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

“…Production, refolding and purification of non-glycosylated rhBMP-2 Non-glycosylated rhBMP-2 was obtained by refolding from E. coli BL21(DE3):pET29c-rhBMP-2 derived inclusion bodies (IBs) and subsequent purification as described previously [9,10,23]. Briefly, solubilization of 1 g of wet rhBMP-2 IBs in 15 ml 6 M guanidine-HCl (Gdn-HCl), 100 mM DTT, 5 mM EDTA, 100 mM tris(hydroxymethyl)aminomethane (TRIS, pH 8.5) was carried out overnight.…”

“…Production of rhBMP-2 through refolding of E. coli based inclusion bodies has been developed long ago as a viable alternative and a more economic source for biologically active rhBMP-2, e.g. [9,10]. Interestingly, E. coli derived rhBMP-2 induces ectopic bone formation at lower concentrations compared to the cell culture derived protein, maybe due to the lack of glycosylation [11].…”

Solubilization and renaturation of biologically active human bone morphogenetic protein-4 from inclusion bodies

“…The protein was produced in the form of inclusion bodies and had to be solubilized and refolded by finding suitable renaturation buffer conditions. Mild solubilization without refolding wasn’t attempted, because studies suggest that cystine knot proteins cannot be obtained in an active form in this manner [ 26 ]. Therefore, different compositions of refolding buffers were tested for the achievement of optimal renaturation yield.…”