Comparison of four methods for the purification and refolding of human interleukin‐2–mouse granulocyte/macrophage colony‐stimulating factor fusion protein

Abstract

The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.

Fusion cytokine IL-2-GMCSF enhances anticancer immune responses through promoting cell–cell interactions

“…1 c, left and middle panels). The specific activities of this fusion cytokine were 3.6 × 10 6 IU/mg for IL-2 and 1.1 × 10 7 IU/mg for GM-CSF respectively, consistent with the results in our previous study [ 34 ] (Fig. 1 c, right panel).…”

“…Fusion protein IL2-GMCSF was prepared [ 34 ] and conserved in our lab. For detection the activity of IL2-GMCSF, CTLL-2 cells or FDC-P1 cells were cultured in the 96-well microplate (Nunc, Thermo Fisher Scientific, Waltham, MA) with serially diluted IL2-GMCSF for 48 h (for IL-2) or 96 h (for GM-CSF).…”

“…To achieve predictable therapeutic effects with combined use of the two potent immunocompetent cytokines, we constructed a IL2-GMCSF fusion cytokine [ 34 ]. Such a fusion cytokine has been reported to enhance anti-tumor immune responses and NK cell activities.…”

Enhanced Detection of Autoantibodies on Protein Microarrays Using a Modified Protein Digestion Technique

“…However, dilution of the protein would not work in this strategy since, prior to printing the proteins, the sample is dried and resuspended in a smaller volume which would reconcentrate them again and cause the proteins to reaggregate into a nonactive state. The more sophisticated chromatographic approaches, on the other hand, vary depending on the protein being renatured. – In our strategy where multiple proteins are being arrayed on a single slide, such a renaturation approach would be difficult and not feasible given the low sample amounts used. Therefore, continuous denaturing conditions were applied to ensure that the protein was not renaturing into a biologically irrelevant conformation or aggregating into larger clusters so that the binding motif was continuously exposed to the serum during hybridization.…”

“…The more sophisticated chromatographic approaches, on the other hand, vary depending on the protein being renatured. [23][24][25][26] In our strategy where multiple proteins are being arrayed on a single slide, such a renaturation approach would be difficult and not feasible given the low sample amounts used. Therefore, continuous denaturing conditions were applied to ensure that the protein was not renaturing into a biologically irrelevant conformation or aggregating into larger clusters so that the binding motif was continuously exposed to the serum during hybridization.…”