Biotechnology and Bioprocess Engineering 2018; 23(6): 670-678  
An Efficient and Rapid Two-step Purification Method for Active Human Macrophage Colony-stimulating Factor from Escherichia coli
Jisu Lee , Jeongmin Lee , Eunha Hwang, Hye-Jung Kim, Sheunghun Lee, Hyerim Lee, Seong Yun Hwang, Sung-Jin Cho, Sun-Ju Yi, and Kyunghwan Kim*
School of Biological Sciences, College of Natural Sciences, Chungbuk National University, Cheongju 28644, Korea
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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.
Keywords: M-CSF, refolding, osteoclastogenesis, purification


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