RESUMO
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.
Assuntos
Biotina/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteínas Recombinantes de Fusão/genética , Estreptavidina/metabolismo , Sequência de Aminoácidos , Animais , Biotina/genética , Biotinilação , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Análise Fatorial , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Camundongos , Células PC-3 , Projetos Piloto , Desnaturação Proteica , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Estreptavidina/genéticaRESUMO
Objective To detect the prokaryotic expression of streptavidin-complement 3d (SA-C3d) fusion protein and verify its function in vitro. Methods The C3d DNA was amplified using C3 cDNA as a template, and the C3d fragment was ligated with the vector plasmid pET-24a-6His-SA-IL15 after the digestion with a one-step cloning method to obtain the SA-C3d prokaryotic expression plasmid. The correctly sequenced plasmid was transformed into expression competent Rosetta to induce protein expression. The target protein was obtained by nickel column affinity chromatography and urea dialyzed refolding. The function of SA was demonstrated by anchoring the biotinylated MB49 cell experiment, and the function of C3d was detected by an experiment that promoted the growth of Raji cells. Results The prokaryotic expression vector of SA-C3d was successfully constructed. The purified target protein was obtained by nickel column purification and dialysis refolding. The protein was specifically bound to biotinylated MB49 cells, which promoted the proliferation of Raji cells in a dose-dependent manner, indicating that the protein SA-C3d had a bifunctional activity. Conclusion The successfully prepared SA-C3d fusion protein can be bound to biotinylated MB49 cells in vitro and promote Raji cell proliferation.
