Abstract
Fusokines are chimeric proteins generated by the physical coupling of cytokines in a single polypeptide, resulting in proteins with highly pleiotropic activity and the potential to treat cancer and autoimmune ailments. For instance, the fusokine GIFT15 (GM-CSF and Interleukin 15 Fusion Transgene) has been shown to be a powerful immunosuppressive protein able to convert naïve B cells into IL-10-producing B cells. To date, the mammalian cell systems used for the expression of GIFT15 allow for secretion of the protein in the culturing media, an inefficient system for producing GMP-compliant fusokines. In this study we report the bacterial expression of bioactive recombinant GIFT15 (rGIFT15). Indeed, there is a constant demand to improve the expression systems for therapeutic proteins. Expression of a maltose-binding protein (MBP) fusion protein efficiently allowed the accumulation of soluble protein in the intracellular milieu. Optimizing the bacterial culture significantly increased the yield of recombinant protein. The biological activity of rGIFT15 was comparable to that of fusokine derived from a mammalian source. This approach led to the production of soluble, endotoxin-free functional protein, averaging 5 mg of rGIFT15 per liter of culture. This process is amenable to scale up for the development of Food and Drug Administration (FDA)-compliant immune-modulatory rGIFT15.
Highlights
Recombinant human cytokines such as G-CSF and IL-2 are routinely used as pharmaceuticals to restore or augment the immune system and combination cytokine therapies have been tested as cancer treatments [1,2]
In order to improve protein yield, the gene encoding GIFT15 was subcloned into pET16b (Novagen) vector that allows for expression of the protein as an N-terminal extension of a His-tag
Gene fusion is an approach that has been successfully used for producing soluble heterologous proteins in E. coli [28], the GIFT15 gene was subcloned in three different vectors that combine the expression of GIFT15 with thioredoxin (TRX), maltose-binding protein (MBP) or glutathione S-transferase (GST) (Fig. 1)
Summary
Recombinant human cytokines such as G-CSF and IL-2 are routinely used as pharmaceuticals to restore or augment the immune system and combination cytokine therapies have been tested as cancer treatments [1,2]. We have developed a novel engineered biopharmaceutical platform – fusokines – for cell-base immune therapy. Fusokines are the fusion of two distinct cytokines in a single polypeptide that results in synergistic bioactivity not observed by the use of parent cytokines [3,4,5,6,7]. The fusion of GM-CSF with CC-chemokine MCP3 (hereafter GMME3) generates a protein that allows for conversion of naıve B-cells to a novel suppressor phenotype [8]
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