Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein.
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.
Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, 138-736, South Korea.
Thu Trang Thi
TT
Vu
Bon-Kyung
BK
Koo
Jung-A
JA
Song
Seon-Ha
SH
Chong
Cho Rong
CR
Park
Minh Tan
MT
Nguyen
Boram
B
Jeong
Han-Bong
HB
Ryu
Jae Young
JY
Seong
Yeon Jin
YJ
Jang
Robert Charles
RC
Robinson
Han
H
Choe
D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D058619:Maltose-Binding Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018932:Chemokine CCL2; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins