Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration

Abstract

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.

The Prodomain of BMP-7 Targets the BMP-7 Complex to the Extracellular Matrix

“…In addition, we showed that acidic pH either dissociates the BMP-7 complex or subjects it to gross conformational changes. Refolding experiments of growth factor domains of other TGF␤ superfamily members have demonstrated the need for harsh denaturants and aggregation suppressors (28) and have suggested that a principal function of the prodomain may be to solubilize the growth factor dimer by shielding the pronounced hydrophobic surfaces present on the mature were separated by SDS-PAGE with (ϩ) or without (Ϫ) disulfide bond reducing agent and transferred to nitrocellulose paper. Anti-histidine (anti-HIS), mAb 2, and mAb 33 immunoblot the prodomain of BMP-7 (ૺ).…”

Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein‐2 with a collagen binding domain derived from von Willebrand factor propolypeptide

“…The method of producing active rhBMP-2 was well explored, 26,34 and was used in our experiments with slight modification. The fused (His) 6 tag could facilitate the protein purification and provide a simple detective method for the protein in the collagen binding assay.…”

“…The two genes were cloned from PCR products, inserted into a high-performance bacterial expression vector pET-28a (Novagen), and transformed into the BL21 (DE3) strain of E. coli. Proteins were prepared as described 26,34 with slight modification. Briefly, the proteins expression was induced with 1 mM isopropyl b-D-thiogalactopyranoside (IPTG) at 378C for 4 h. The recombinant fusion protein was isolated from Escherichia coli as inclusion bodies by sonication and centrifugation, and then dissolved with 8M urea and purified using nickel chelate chromatography (Amersham Biosciences).…”

“…Purity and yields of recombinant proteins were analyzed by electrophoresis on a 15% SDS-PAGE. The purified proteins were renatured as described, 26 then they were concentrated to 2 mg/mL by ultrafiltration (Amicon Ultra-15 Centrifugal Filter Devices, Millipore), and stored at À808C.…”

“…To enhance the binding of BMP-2 to DBM and to prevent the release of the BMP-2 to the surrounding environment, a collagen binding domain (CBD) 25,26 was added to the N-terminal of the growth factor of BMP-2. This peptide was derived from the D2 collagen-targeting domain for von Willebrand factor (vWF) propolypeptide.…”