The pilot-scale preparation of the SA-hGM-CSF bi-functional fusion protein

Bioengineered. 2019 Dec;10(1):108-120. doi: 10.1080/21655979.2019.1608712.

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.

Keywords: SA-hGM-CSF fusion protein; optimization; pilot-scale; purification; refolding.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biotin / genetics
  • Biotin / metabolism*
  • Biotinylation
  • Cell Line, Tumor
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Factor Analysis, Statistical
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics*
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Humans
  • Mice
  • PC-3 Cells
  • Pilot Projects
  • Protein Denaturation
  • Protein Refolding
  • Protein Stability
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification
  • Streptavidin / genetics
  • Streptavidin / metabolism*

Substances

  • Recombinant Fusion Proteins
  • Biotin
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Streptavidin

Grant support

This work was supported by the National Natural Science Foundation of China [81573110];National Natural Science Foundation of China [91729101].