title: Maltose-binding protein fusion allows for high level bacterial expression and purification of bioactive mammalian cytokine derivatives.
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.
authors.affiliation: Department of Hematology and Medical Oncology, Emory University, Winship Cancer Institute, Atlanta, Georgia, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pennati
authors.affiliation: Department of Hematology and Medical Oncology, Emory University, Winship Cancer Institute, Atlanta, Georgia, United States of America.
authors.forename: Jiusheng
authors.initials: J
authors.lastname: Deng
authors.affiliation: Department of Hematology and Medical Oncology, Emory University, Winship Cancer Institute, Atlanta, Georgia, United States of America; Department of Pediatrics, Emory University, Winship Cancer Institute, Atlanta, Georgia, United States of America.
authors.forename: Jacques
authors.initials: J
authors.lastname: Galipeau
mesh_terms: D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D015153:Blotting, Western; D016207:Cytokines / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D058619:Maltose-Binding Proteins / Q000235:genetics*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D016207:Cytokines; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins