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Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein

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Abstract

Human chemokine (C–C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags—Trx, GST, MBP, NusA, His8, PDI, and PDIb′a′—for their ability to allow the soluble overexpression of hCCL2. Three tags—MBP, His8, and PDI—solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.

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Acknowledgments

This work was supported by the Priority Research Center Program (2009-0094050) and the Korea Research Foundation (2010-0029522) funded by the Ministry of Education, Science and Technology, Korea.

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Authors and Affiliations

  1. Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, 138-736, South Korea

    Thu Trang Thi Vu, Bon-Kyung Koo, Jung-A Song, Seon-Ha Chong, Minh Tan Nguyen, Boram Jeong, Han-Bong Ryu, Yeon Jin Jang & Han Choe

  2. Laboratory of G-protein-Coupled Receptors, Graduate School of Medicine, Korea University, Seoul, 136-705, South Korea

    Cho Rong Park & Jae Young Seong

  3. Institute of Molecular and Cell Biology, A*STAR (Agency of Science, Technology and Research), Biopolis, Singapore, 138673, Singapore

    Robert Charles Robinson

  4. Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore, 117597, Singapore

    Robert Charles Robinson

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  1. Thu Trang Thi Vu
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Correspondence to Han Choe.

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Thu Trang Thi Vu and Bon-Kyung Koo have contributed equally to this work.

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Vu, T.T.T., Koo, BK., Song, JA. et al. Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein. Mol Biol Rep 42, 651–663 (2015). https://doi.org/10.1007/s11033-014-3812-3

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