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

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.

Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF

“…Fusion technology with tags such as MBP, N-utilization substance protein A, or protein disulfide bond isomerase has been shown to be effective for enhancing protein expression as well as solubility 16, 19 . Because of its relatively small size, easy purification using MBP chromatography, and efficacy in supporting the expression as well as proper folding of target proteins in the reducing environment of E. coli cytoplasm, MBP is largely the tag of choice.…”

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag

“…Based on the protein expression level, solubility, and size of the fusion protein, we selected the MBP-tagged construct for further purification. The MBP tag has been demonstrated to assist the soluble expression and purification of bioactive hCCL2, crotamine, hGCSF, and hEC-SOD with a high yield and high purity [Bae et al, 2013;Do et al, 2014;Vu et al, 2014Vu et al, , 2015. MBP can promote the proper folding of the attached protein into its biologically active conformation [Kapust and Waugh, 1999].…”

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

“…To this end, one of eight tags – His6, Sumo, Trx, GST, PDIb′a′, MBP, NusA, or PDI – was attached to the N terminus of hFGF21, resulting in eight recombinant vectors. Previously, these tags have been used in multiple studies to improve the soluble expression of various recombinant proteins in E. coli 19 – 23 , 27 , 28 . In the present study, the hFGF21 fusion vectors were transformed into the E. coli BL21(DE3) to characterise the behaviour of the fusion proteins expressed in the cytoplasm.…”