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KMID : 1094720180230060670
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Biotechnology and Bioprocess Engineering
2018 Volume.23 No. 6 p.670 ~ p.678
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An Efficient and Rapid Two-step Purification Method for Active Human Macrophage Colony-stimulating Factor from Escherichia coli
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Lee Ji-Su
Lee Jeong-Min
Hwang Eun-Ha
Kim Hye-Jung
Lee Sheung-Hun
Lee Hye-Rim
Hwang Seong-Yun
Cho Sung-Jin
Yi Sun-Ju
Kim Kyung-Hwan
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Abstract
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Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of mononuclear phagocytes. To be biologically active, M-CSF must form a homodimer linked by disulfide bridges. However, the refolding step of recombinant M-CSF is not only extremely laborious but also rate-limiting. Here, we describe an efficient and simplified method for refolding and purifying M-CSF from Escherichia coli. A truncated M-CSF (amino acids 36?181) was tagged with histidine, expressed, and then purified using nickel affinity columns under denaturing conditions. Our redox refolding buffer containing a mixture of reduced and oxidized glutathione and arginine correctly refolded M-CSF. Chromatography on Q-sepharose columns selectively purified the M-CSF dimer from the monomeric form. The dimeric nature of the purified M-CSF was confirmed using multi angle light scattering combined with high performance liquid chromatography. Moreover, cell proliferation and osteoclast differentiation assays using bone marrow-derived macrophages demonstrated that the recombinant M-CSF was biologically active ex vivo.
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KEYWORD
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M-CSF, refolding, osteoclastogenesis, purification
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