RESUMEN
The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.
Asunto(s)
Proteínas Anfibias/genética , Antineoplásicos/farmacología , Clonación Molecular/métodos , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Ribonucleasas/genética , Proteínas Anfibias/biosíntesis , Proteínas Anfibias/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Expresión Génica , Humanos , Concentración 50 Inhibidora , Extracción Líquido-Líquido , Pichia/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Ingeniería de Proteínas , Señales de Clasificación de Proteína , Rana pipiens/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Rodaminas/química , Ribonucleasas/biosíntesis , Ribonucleasas/farmacología , Albúmina Sérica/biosíntesis , Albúmina Sérica/genética , Relación Estructura-Actividad , Transformación GenéticaRESUMEN
Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.