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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Improved Soluble Expression in Escherichia coli and Easily Purified Recombinant Human Bone Morphogenetic 7-2 Fusion Protein

Author(s): Shuai Wang, Min Lai, Yaqian Cui, Hongwei Fan* and Kaizong Huang*

Volume 29, Issue 6, 2022

Published on: 24 June, 2022

Page: [550 - 554] Pages: 5

DOI: 10.2174/0929866529666220420114713

Price: $65

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Abstract

Background: Bone morphogenetic protein (BMP) is a cysteine-rich growth factor and plays a key role in early bone tissue development and bone defect repair. However, the low yield, high cost and complicated process in BMP significantly limit its clinical application.

Objective: In this study, we developed an efficient method for soluble expression and preparation of recombinant human bone morphogenetic 7-2 fusion protein (rhBMP7-2) and determined its molecular weight and biological activity.

Methods: The fusion gene for rhBMP-2 and rhBMP-7 was inserted into the pET-ELP expression vector. Correct DNA sequence was confirmed, the rhBMP7-2-ELP was transformed into Escherichia coli strain BL21 (DE3), and the rhBMP7-2 was produced in the recombinant E. coli. Recombinant BMP7-2 purify was identified using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The cell proliferation and biological activity of rhBMP7-2 were measured by Cell Counting Kit-8 and Alkaline Phosphatase assay using C2C12 cells, respectively.

Results: The result of digestion of NdeI, BamHI and XhoI enzymes showed that the rhBMP7-2-ELP was correctly constructed. The recombinant BMP7-2 was successfully expressed in soluble form; the purified rhBMP7-2 showed biological activity and significantly promoted cell proliferation and differentiation in a dose-dependent manner.

Conclusion: The rhBMP7-2 fusion protein with osteogenic activity was prepared through a lowcost and time-efficient method. Our preparation method presents the potential to be applied to the large-scale production of rhBMP7-2 and is expected to play a significant role in clinical treatment.

Keywords: Recombinant human bone morphogenetic protein, Escherichia coli, soluble expression, one step, purification, timeefficient.

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[1]
Urist, M.R. Bone: formation by autoinduction. Science, 1965, 150(3698), 893-899.
[http://dx.doi.org/10.1126/science.150.3698.893] [PMID: 5319761]
[2]
Wall, A.; Board, T. Bone: Formation by autoinduction. Science, 2014, 395(3698), 449-451.
[3]
Wozney; John, M., Overview of bone morphogenetic proteins. Spine, 2002, 27(Suppl.), 2-8.
[http://dx.doi.org/10.1097/00007632-200208151-00002]
[4]
Lowery, J.W.; Rosen, V. The BMP pathway and its inhibitors in the skeleton. Physiol. Rev., 2018, 98(4), 2431-2452.
[http://dx.doi.org/10.1152/physrev.00028.2017] [PMID: 30156494]
[5]
O’hEireamhoin. S.; Quinlan, J.F.; Rourke, K.O. The use of bone morphogenetic protein 7 in fracture non-unions. Orthop. Surg., 2011, 3(1), 40-44.
[http://dx.doi.org/10.1111/j.1757-7861.2010.00119.x] [PMID: 22009979]
[6]
Lissenberg-Thunnissen, S.N.; de Gorter, D.J.; Sier, C.F.; Schipper, I.B. Use and efficacy of bone morphogenetic proteins in fracture healing. Int. Orthop., 2011, 35(9), 1271-1280.
[http://dx.doi.org/10.1007/s00264-011-1301-z] [PMID: 21698428]
[7]
Bessa, P.C.; Casal, M.; Reis, R.L. Bone morphogenetic proteins in tissue engineering: The road from laboratory to clinic, part II (BMP delivery). J. Tissue Eng. Regen. Med., 2008, 2(2-3), 81-96.
[http://dx.doi.org/10.1002/term.74] [PMID: 18383454]
[8]
Koh, J.T.; Zhao, Z.; Wang, Z.; Lewis, I.S.; Krebsbach, P.H.; Franceschi, R.T. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J. Dent. Res., 2008, 87(9), 845-849.
[http://dx.doi.org/10.1177/154405910808700906] [PMID: 18719211]
[9]
Nasrabadi, D.; Rezaeiani, S.; Sayadmanesh, A.; Baghaban Eslaminejad, M.; Shabani, A. Inclusion body expression and refolding of recombinant bone morphogenetic protein-2. Avicenna J. Med. Biotechnol., 2018, 10(4), 202-207.
[PMID: 30555651]
[10]
Huang, K.; Gao, M.; Lin, F.; Lai, Y.; Fan, H.; Hua, Z. IR820 covalently linked with self-assembled polypeptide for photothermal therapy applications in cancer. Biomater. Sci., 2018, 6.
[http://dx.doi.org/10.1039/C8BM00399H]
[11]
Bustos-Valenzuela, J.C.; Halcsik, E.; Bassi, E.J.; Demasi, M.A.; Granjeiro, J.M.; Sogayar, M.C. Expression, purification, bioactivity, and partial characterization of a recombinant human bone morphogenetic protein-7 produced in human 293T cells. Mol. Biotechnol., 2010, 46(2), 118-126.
[http://dx.doi.org/10.1007/s12033-010-9287-0] [PMID: 20499289]
[12]
Zhang, Y.; Ma, Y.; Yang, M.; Min, S.; Yao, J.; Zhu, L. Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro. Protein Expr. Purif., 2011, 75(2), 155-160.
[http://dx.doi.org/10.1016/j.pep.2010.07.014] [PMID: 20691269]
[13]
Franceschi, R.T. Biological approaches to bone regeneration by gene therapy. J. Dent. Res., 2005, 84(12), 1093-1103.
[http://dx.doi.org/10.1177/154405910508401204] [PMID: 16304438]
[14]
Miao, C.; Qin, D.; Cao, P.; Lu, P.; Xia, Y.; Li, M.; Sun, M.; Zhang, W.; Yang, F.; Zhang, Y.; Tang, S.; Liu, T.; Liu, F. BMP2/7 heterodimer enhances osteogenic differentiation of rat BMSCs via ERK signaling compared with respective homodimers. J. Cell. Biochem., 2018.
[http://dx.doi.org/10.1002/jcb.28162] [PMID: 30485526]
[15]
Kirker-Head, C.A. Potential applications and delivery strategies for bone morphogenetic proteins. Adv. Drug Deliv. Rev., 2000, 43(1), 65-92.
[http://dx.doi.org/10.1016/S0169-409X(00)00078-8] [PMID: 10967222]
[16]
Retnoningrum, D.S.; Pramesti, H.T.; Santika, P.Y.; Valerius, O.; Asjarie, S.; Suciati, T. Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli. Protein Expr. Purif., 2012, 84(2), 188-194.
[http://dx.doi.org/10.1016/j.pep.2012.05.010] [PMID: 22691543]
[17]
Wang, G.; Han, J.; Wang, S.; Li, P. Expression and purification of recombinant human bone morphogenetic protein-7 in Escherichia coli. Prep. Biochem. Biotechnol., 2014, 44(1), 16-25.
[http://dx.doi.org/10.1080/10826068.2013.782043] [PMID: 24117149]
[18]
Zhang, H.; Wu, J.; Zhang, Y.; Fu, N.; Wang, J.; Zhao, S. Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations. Mol. Biol. Rep., 2010, 37(7), 3089-3095.
[http://dx.doi.org/10.1007/s11033-009-9883-x] [PMID: 19823948]
[19]
Long, S.; Truong, L.; Bennett, K.; Phillips, A.; Wong-Staal, F.; Ma, H. Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli. Protein Expr. Purif., 2006, 46(2), 374-378.
[http://dx.doi.org/10.1016/j.pep.2005.09.025] [PMID: 16298141]
[20]
Valera, E.; Isaacs, M.J.; Kawakami, Y.; Izpisúa Belmonte, J.C.; Choe, S. BMP-2/6 heterodimer is more effective than BMP-2 or BMP-6 homodimers as inductor of differentiation of human embryonic stem cells. PLoS One, 2010, 5(6)e11167
[http://dx.doi.org/10.1371/journal.pone.0011167] [PMID: 20567515]
[21]
Dang, J.; Jing, L.; Shi, W.; Qin, P.; Li, Y.; Diao, A. Expression and purification of active recombinant human bone morphogenetic 7-2 dimer fusion protein. Protein Expr. Purif., 2015, 115, 61-68.
[http://dx.doi.org/10.1016/j.pep.2015.08.010] [PMID: 26276472]

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