Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing

Abstract

One problem associated with clinical application of CHO-derived recombinant human bone morphogenetic protein (C-BMP-2) is its high cost due to the need for use of high doses. To solve this problem, Escherichia coli-derived BMP-2 (E-BMP-2) has been examined using the technique of molecular unfolding and refolding. However, it is unclear whether the characteristics of E-BMP-2 are appropriate for clinical application. In this study, we examined the biological activity of E-BMP-2 and its heat tolerance in in vitro and in vivo systems. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the high purity of E-BMP-2. E-BMP-2-induced alkaline phosphatase expression in osteoprogenitor cells (C2C12, ST2, and primary murine calvarial osteoblast cells) was dose-dependent, and consistently elicited ectopic new ossicles of significant size in mice, also in dose-dependent fashion. In addition, E-BMP-2 induced phosphorylation of Smad1/5/8 and mRNA expression of osteoblastic differentiation markers to the same extent as C-BMP-2. On the other hand, when E-BMP-2 was exposed to increasing heat over time, its bone-inducing capacity was maintained until reaching 70 degrees C for 2 h or 90 degrees C for 15 min. Thus, E-BMP-2 will exhibit a decrease in activity with the sterilization procedures required prior to use in surgery. These findings indicate that the biological capacity and heat stability of E-BMP-2 are almost equivalent to those of currently available C-BMP-2, and suggest that E-BMP-2 might, thus, solve current problems of cost impeding routine clinical use of rhBMP-2.

Effect of Escherichia coli-produced recombinant human bone morphogenetic protein 2 on the regeneration of canine segmental ulnar defects

“…In contrast to CHO-BMP-2, E-BMP-2 is not glycosylated, but previous in vitro studies suggested that its biological activity is similar to that of CHO-BMP-2 [22,23]. Thus, the bacterial expression system appears to offer a viable method for achieving an extremely high protein output at a relatively low cost [21].…”

Successful Spinal Fusion by E. coli-derived BMP-2-adsorbed Porous β-TCP Granules: A Pilot Study

“…Third, our sample size in each group was not sufficient to yield substantial effects and the study is likely underpowered; we therefore consider the study preliminary. On the other hand, we previously found E-BMP-2 has osteoinductive activity equivalent to that currently produced in BMP-2 gene-transfected animal cells (Chinese hamster ovary cells) [45]. However, it is essential to evaluate the safety of E-BMP-2 for clinical use.…”

“…However, it is essential to evaluate the safety of E-BMP-2 for clinical use. In several previous studies using E-BMP-2 [18,29,45], its safety was not sufficiently examined. Evaluation of the safety of E-BMP-2 is required in preclinical studies.…”

“…To address this issue, Sebald et al devised a novel method to produce rhBMP-2 derived from E. coli and convert BMP monomers to biologically active dimers (E-BMP-2) [18,29]. E-BMP-2 has osteoinductive activity the same as CHO-derived rhBMP-2 both in vivo and in vitro [45]. However, these reports included only results for E-BMP-2-induced ectopic bone formation.…”

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

“…Initially, BMPs were isolated directly from bones but the yields were low and the process was labourious (Bessho et al 1989;Hu et al 2004). Recombinant human BMPs (rhBMPs) were produced from Escherichia coli (Hillger et al 2005;Yano et al 2009) and mammalian cell culture systems (Israel et al 1992;Wang et al 1990;Yamaguchi et al 1991). Despite successfully producing properly folded, glycosylated and biologically active rhBMP-2, the yield of mammalian cell expression system is low, that remains a major challenge and hinders the use of rhBMP-2 in therapeutic applications.…”