CHIANG MAI UNIVERSITY JOURNAL OF NATURAL SCIENCES

Volume 19, No. 04, Month OCTOBER, Year 2020, Pages 752 - 773

Soluble expression and purification of bioactive recombinant human bone morphogenetic protein-2 from escherichia coli

Waraporn Kasekarn, Benjawan Suksiriphattanapong, Tawan Chokepaichitkool, Orawan Wanachewin, Sittiruk Roytrakul, Prachya Kongtawelert

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Human bone morphogenetic protein-2 (hBMP-2) is a potent growth and differentiation factor for bone induction and regeneration. Recombinant hBMP-2 (rhBMP-2) was cloned and expressed as a soluble protein using E. coli-based expression system. A full-length gene encoding mature hBMP-2 was amplified by RT-PCR, cloned into an expression vector and expressed using SHuffle E. coli cells. The rhBMP-2 was successfully expressed as a soluble protein under the control of the lacUV5 and protein A promoters by IPTG induction. The rhBMP-2 fused with ZZ domain at its N-terminus was successively purified with a single step by using IgG Sepharose 6 fast flow affinity chromatography. Analysis of the purified protein on SDS-PAGE, Western blot analysis and LC-MS/MS, verified that the purified protein was rhBMP-2. The biological activity testing on hFOB 1.19 showed that rhBMP-2 had the ability to significantly induce cell proliferation in a dose dependent manner. ALP staining and activity assay also increased after rhBMP-2 treatment. The mRNA expression of the osteogenic genes by quantitative real-time PCR (qRT-PCR) showed that rhBMP-2 was able to up-regulate the gene expression of ALP, COL1, BMP-2, Runx2, and OPN. This data indicates that rhBMP-2 is functionally active to induce human osteoblast proliferation and differentiation. The production of rhBMP-2 by this developed method could be useful for bone regeneration and repair applications.

Keywords

Human bone morphogenetic protein-2, Recombinant protein, Soluble protein, Osteoblast differentiation, Escherichia coli

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