Development of Refolding Process to Obtain Active Recombinant Human Bone Morphogenetic Protein-2 and its Osteogenic Efficacy on Oral Stem Cells
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BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED50 of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.
Abstract
BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED50 of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.
참고문헌 (30)
- Amitabha Bandyopadhyay, "BMP signaling in development and diseases: A pharmacological perspective", Biochemical Pharmacology, 85(7), pp.857~864 (2013), Elsevier BV[DOI]
- Shah NM, "Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members", Cell, 85, pp.331~343 (1996)
- Sandrine Pizette, "BMPs Are Required at Two Steps of Limb Chondrogenesis: Formation of Prechondrogenic Condensations and Their Differentiation into Chondrocytes", Developmental Biology, 219(2), pp.237~249 (2000), Elsevier BV[DOI]
- Bettina Keller, "Interaction of TGFβ and BMP Signaling Pathways during Chondrogenesis", PLoS ONE, 6(1), pp.e16421~ (2011), Public Library of Science (PLoS)[DOI]
- Zou H, "Requirement for BMP signaling in interdigital apoptosis and scale formation", Science, 272, pp.738~741 (1996)
- Amitabha Bandyopadhyay, "Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis", PLoS Genetics, 2(12), pp.e216~ (2006), Public Library of Science (PLoS)[DOI]
- MICHAEL CENTRELLA, "Transforming Growth Factor-β Gene Family Members and Bone*", Endocrine Reviews, 15(1), pp.27~39 (1994), The Endocrine Society[DOI]
- Wozney JM, "Novel regulators of bone formation : molecular clones and activities", Science, 242, pp.1528~1534 (1988)
- Steven K Pregizer, "Dynamics and cellular localization of Bmp2, Bmp4, and Noggin transcription in the postnatal mouse skeleton", Journal of Bone and Mineral Research, 30(1), pp.64~70 (2015), Wiley[DOI]
- T Katagiri, "Bone Morphogenetic Protein-2 Inhibits Terminal Differentiation of Myogenic Cells by Suppressing the Transcriptional Activity of MyoD and Myogenin", Experimental Cell Research, 230(2), pp.342~351 (1997), Elsevier BV[DOI]
- Katagiri T, "Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage", J Cell Biol, 127, pp.1755~1766 (1994)
- Massague J, "Controlling TGF-beta signaling", Genes Dev, 14, pp.627~644 (2000)
- Massague J, "Transcriptional control by the TGFbeta", Smad signaling system, 2000, pp.19, 1745~1754
- P. C. Bessa, "Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery)", Journal of Tissue Engineering and Regenerative Medicine, 2(2-3), pp.81~96 (2008), Wiley[DOI]
- William F. McKay, "A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE® Bone Graft)", International Orthopaedics, 31(6), pp.729~734 (2007), Springer Nature[DOI]
- Wang EA, "Recombinant human bone morphogenetic protein induces bone formation", Proc Natl Acad Sci U S A, 87, pp.2220~2224 (1990)
- Israel DI, "Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells", Growth Factors, 7, pp.139~150 (1992)
- Vallejo LF, "Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli", J Biotechnol, 94, pp.185~194 (2002)
- Hyo Jin Ihm, "Soluble expression and purification of synthetic human bone morphogenetic protein-2in Escherichia coli", BMB reports, 41(5), pp.404~407 (2008), 한국생화학분자생물학회
- JUNG-KWON CHOI, "The efficiency of the in vitro osteo", dentinogenic differentiation of human dental pulp cells, periodontal ligament cells and gingival fibroblasts, 2015, pp.35(1), 161~168[DOI]
- Jin-Hee Min, "Dentinogenic potential of human adult dental pulp cells during the extended primary culture", Human Cell, 24(1), pp.43~50 (2011), Springer Nature[DOI]
- R.H. Khan, "Solubilization of Recombinant Ovine Growth Hormone with Retention of Native-like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli", Biotechnology Progress, 14(5), pp.722~728 (1998), Wiley[DOI]
- Surinder Mohan Singh, "Solubilization and refolding of bacterial inclusion body proteins", Journal of Bioscience and Bioengineering, 99(4), pp.303~310 (2005), Elsevier BV[DOI]
- St John RJ, "High pressure fosters protein refolding from aggregates at high concentrations", Proc Natl Acad Sci U S A, 96, pp.13029~13033 (1999)
- Burgess RR, "Purification of overproduced Escherichia coli RNA polymerase sigma factors by solubilizing inclusion bodies and refolding from Sarkosyl", Methods Enzymol, 273, pp.145~149 (1996)
- Motonori Kudou, "Refolding single-chain antibody (scFv) using lauroyl-l-glutamate as a solubilization detergent and arginine as a refolding additive", Protein Expression and Purification, 77(1), pp.68~74 (2011), Elsevier BV[DOI]
- Surinder M. Singh, "Solubilization of inclusion body proteins using n -propanol and its refolding into bioactive form", Protein Expression and Purification, 81(1), pp.75~82 (2012), Elsevier BV[DOI]
- Arun K. Upadhyay, "Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein", Frontiers in Microbiology, 5, pp.486~ (2014), Frontiers Media SA[DOI]
- Simona Jevševar, "Production of Nonclassical Inclusion Bodies from Which Correctly Folded Protein Can Be Extracted", Biotechnology Progress, 21(2), pp.632~639 (2005), Wiley[DOI]
- Shinong Long, "Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli", Protein Expression and Purification, 46(2), pp.374~378 (2006), Elsevier BV[DOI]
