Expression and Purification of Biologically Active Rat Bone Morphogenetic Protein-4 produced as Inclusion Bodies in Recombinant Escherichia coli

Abstract

Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mM) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.

Osteoinduction in human fat-derived stem cells by recombinant human bone morphogenetic protein-2 produced in Escherichia coli

“…In previous studies, recombinant BMPs obtained from E. coli were tested in murine cells lines C2C12 (Vallejo et al 2002;Long et al 2006), MC3T3-E1 (Klösch et al 2005) and CH310T1/2 (Yang et al 2003) or in vivo rodent models (Kubler et al 1998;Bessho et al 2000;Hillger et al 2005). In our case, bioactivity was achieved using primary cultures of human stem cells.…”

“…Runx2/Cbfa1 OSX Smad-5 Klösch et al (2005) and Long et al (2006). Formation of cysteine bridges is favored at slightly alkaline pH, thus increasing amounts of dimeric rhBMP-2 (Vallejo and Rinas 2004).…”

Recombinant BMP 4/7 fusion protein induces differentiation of bone marrow stem cells

“…Although all BMPs are glycosylated in vivo, their osteoinductive activity is not dependent on glycosylation [Elima, 1993]. Therefore, prokaryotic expression systems have been extensively used for large-scale expression of BMP4 [Klosch et al, 2005] and BMP2 [Ihm et al, 2008;Zhang et al, 2010]. Recombinant BMP2 and BMP7 have been approved for human trials in a number of countries, making it imperative to optimize large-scale production of these proteins [Rutherford et al, 2003].…”

“…In order to advance the efforts to optimize protein therapy strategies for BMP-mediated bone regeneration, a number of groups have successfully used prokaryotic expression systems for large-scale production of biologically active, recombinant BMPs [Klosch et al, 2005;Ihm et al, 2008;Zhang et al, 2010]. In this study, we investigated the feasibility of using Escherichia coli-derived human recombinant BMP4/7 fusion protein to induce differentiation of BMSCs.…”

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

“…Such biological requirements for production of these proteins demanded the use of mammalian cell expression systems, such as CHO, BSC-1 [10], and COS7 [11] cells lines, as previously reported. Prokaryotic systems (Escherichia coli) were also used for the production of BMPs [12][13][14][15], but the refolding and assembly steps, which are necessary to obtain considerable amounts of the protein with biological activity, do not encourage scale-up processes.…”