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.
Bibliographical noteFunding Information:
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.
© 2014, Springer Science+Business Media Dordrecht.
- Chemotatic migration assay
- Human chemokine (C–C motif) ligand 2
- Protein purification from E. coli
- Soluble overexpression
ASJC Scopus subject areas
- Molecular Biology