E. coli 발현 시스템에 의해 생산된 recombinant human bone morphogenetic protein-2의 정제와 생물학적 활성
Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system
2
조회
0
다운로드
7
인용
0
참고문헌
초록
Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell.Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells. (J Korean Acad Periodontol 2008;38:41-50)
Abstract
Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell.Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells. (J Korean Acad Periodontol 2008;38:41-50)
참고문헌 (36)
- Urist MR, "Bone Formation by autoinduction", Science, 150, pp.893~899 (1965)
- Wozney JM, "Novel regulators of bone formation: molecular clones and activities", Scien, 242, pp.1528~1534 (1988)
- Cunningham NS, "Osteogennin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor β1 mRNA expression", Proc Natl Acad Sci USA, 89, pp.11740~11744 (1992)
- Paralkar VM, "Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation and binding to type IV collagen", J Cell Biol, 119, pp.1721~1728 (1992)
- Hogan BLM, "Bone morphogenetic proteins: multifunctional regulators of vertebrate development", Genes Dev, 10, pp.1580~1594 (1996)
- Barnes GL, "Growth factor regulation of fracture repair", J Bone Miner Res, 14, pp.1805~1815 (1999)
- Wikesjö UM, "Periodontal repair in dogs: effect of rhBMP-2 concentration on regeneration of alveolar bone and periodontal attach- ment", J Clin Periodontol, 26, pp.392~400 (1999)
- Kim CS, "The effect of recombinant human bone morphogenetic protein-4 on the osteoblastic differentiation of mouse calvarial cells affected by Porphyromonas gingivalis", J Periodontol, 73, pp.1126~1132 (2002)
- Kingsley DM, "The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGF beta superfamily", Cell, 71, pp.399~410 (1992)
- Solloway MJ, "Mice lacking Bmp6 function", Dev Genet, 22, pp.321~339 (1998)
- Jena N, "BMP7 null mutation in mice: developmental defects in skeleton, kidney, and eye", Exp Cell Res, 230, pp.28~37 (1997)
- Wang EA, "Recombinant human bone morphogenetic protein induces bone formation", Proc Natl Acad Sci USA, 87, pp.2220~2224 (1990)
- Chen D, "Bone morphogenetic proteins", Growth Factors, 22, pp.233~241 (2004)
- Cook SD, "Evaluation of recombinant human osteogenic protein-1(rhOP-1) placed with dental implant in fresh extraction sites", J Oral Implantol, 21, pp.281~289 (1995)
- Cook SD, "Effects of recombinant human osteogenic protein-1 on healing of segmental defects in non-human primates", J Bone Joint Surg Am, 77, pp.734~750 (1995)
- Jones AL, "Recombinant human BMP-2 and allograft compared with autogenous bone graft for reconstruction of diaphyseal tibial fractures with cortical defects: a randomized, controlled trial", J Bone Joint Surg Am, 88, pp.1431~1441 (2006)
- Govender S, "Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty paients", J Bone Joint Surg Am, 84, pp.2123~2134 (2002)
- Wang EA, "Recombinant human bone morphogenetic protein induces bone formation", Proc Natl Acad Sci USA, 87, pp.2220~2224 (1990)
- Sampath TK, "Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro", J Biol Chem, 267, pp.20352~20362 (1992)
- Maruoka Y, "Production of functional human bone morphogenetic protein -2 using a baculovirus", Sf-9 insect cell system, 1995, pp.35, 957~963
- Hazama M, "Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system", Biochem Biophys Res Commun, 209, pp.859~866 (1995)
- Kubler NR, "Inductive properties of recombinant human BMP-2 produced in a bacterial expression system", Int J Oral Maxiilofac Surg, 27, pp.305~399 (1998)
- Vallejo LF, "Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density clutures of recombinant Escherichia coli", J Biotech, 94, pp.185~194 (2002)
- Ruppert R, "Human bone morphogenetic protein-2 contains a heparin-binding sites which modifies its biological activity", Eur J Biochem, 237, pp.295~302 (1996)
- Tabandeh F, "Heat-induced production of human growth hormone by high cell density cultivation of recombinant Escherichia coli", Biotechnol Lett, 26, pp.245~250 (2004)
- Korz DJ, "Simple fed-batch technique for high cell density cultivation of Escherichia coli", J of Biotechnology, 39, pp.59~65 (1995)
- Katagiri T, "The non-osteogenic mouse pluripotent cell line C3H10T1", 2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic proteins-2, 1990, pp.172, 295~299
- Anderson HC, "The mechanismof bone induction and bone healing by human osteosarcoma cell extracts", Clin Orthop Relat Res, 313, pp.129~134 (1995)
- Hoffmann A, "Perspectives in the biological function, the technical and therapeutic application of bone morphogenetic proteins", Appl Microbiol Biotechnol, 57, pp.294~308 (2001)
- Long S, "Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli", Protein Expr Purif, 46, pp.374~378 (2006)
- Kato M, "Optimized use of a biodegradeble polymer as a carrier material for the local delivery of recombinant human bone morphogenetic protein-2(rhBMP-2)", Biomaterials, 27, pp.2035~2041 (2006)
- Gimble JM, "Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells", J Cell Biochem, 58, pp.393~402 (1995)
- Koempel JA, "The effect of recombinant human bone morphogenetic protein-2 on the integration of porous hydroxyapatite implants with bone", J Biomed Mater Res, 41, pp.359~363 (1988)
- Hong S-J, "The effects of a fibrin-fibronectin", β-tricalcium phosphate, Biomaterials, pp.2006, 27[DOI]
- Kim C-S, "Ectopic bone formation associated with recombinant human bone morphogenetic proteins-2 using absorbable collagen sponge and beta tricalcium phosphate as carriers", Biomaterials, 26, pp.2501~2507 (2005)
- 송대석, "재조합 골형성 단백질 2형(rh-BMP-2) 함유 무세포성 진피 조직(acellualr dermal matrix)의 골재생 효과", 대한치주과학회지, 37(3), pp.511~522 (2007), 대한치주과학회
