Abstract
ABSTRACT The granulocyte-macrophage colony-stimulating factor (GM-CSF) can be used to induce a powerful immune response. Based on the specific binding of biotin and streptavidin, SA-hGM-CSF was anchored on the surface of biotinylated tumor cells, which could enhance the anti-tumor effect of tumor cell vaccines in our previous reports, suggesting it would have potential clinical value. Preparation of the biologically active proteins in large-scale production is the basis of clinical application, however, only a small amount of biologically active protein was obtained according to previous studies. In this study, we researched the effects of various factors on the purification and simultaneous renaturation of SA-hGM-CSF fusion protein by single factor experiment and orthogonal experiment. Here, we developed a viable pilot-scale trial in the fermentation, purification, refolding and freeze-drying of SA-hGM-CSF proteins in order to efficiently obtain more biologically active proteins with high purity, which will lay the foundation for industrial production.
Highlights
Granulocyte macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor and immune protein which can be used in vaccines and clinical treatment [1]
Effect of the single factor on purification and simultaneous refolding of the SA-hGM-CSF fusion protein Based on the DEAE sepharose Fast Flow ion exchange chromatography, various factors affecting purification, biological activity, and recovery of renatured proteins need to be determined, including pH of refolding and elution buffer (Figure 2(a), Fig. S2A and B), urea gradient (Figure 2(b), Fig. S2C and D), sample flow velocity(Figure 2(c), Fig. S2E and F), the sample load of the SA-hGM-CSF (Figure 2(d), Figure S2G and H), the GSH/GSSG ratio of refolding buffer (Figure 2(e), Figure S2I and J), urea concentrate of refolding buffer (Figure 2(f), Fig. S2K and L), and the amount of L-arginine in the refolding buffer (Figure 2(g), Fig. S2M and N)
The studies in this paper focused on exploring and optimizing the conditions for the large-scale production of biologically active proteins
Summary
Granulocyte macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor and immune protein which can be used in vaccines and clinical treatment [1]. It contains 127 amino acid proteins formed by the antiparallel arrangement of the two β-sheet and four α-helix bundles [2]. There is no omnipotent method to facilitate the purification simultaneous renaturation of any given protein. It is necessary to explore and optimize the method for the purification simultaneous renaturation of SA-hGM-CSF to make it more effective and simplified. We have demonstrated experimental procedures for the production of SA-hGM-CSF protein on a pilot scale, which has been explored and optimized through fermentation, purification, and renaturation. The highly efficient and timesaving pilot-scale production of SA-hGM-CSF
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