The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P GCW14 , and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P GCW14 were cloned upstream of the EGFP gene and promoter activity was analyzed by measuring fluorescence intensity. P GCW14 exhibited significantly stronger promoter activity than the classic strong constitutive promoters P TEF1 and P GAP under various carbon sources, suggesting that P GCW14 is a strong and constitutive promoter. Hence, P GCW14 can be used as a promoter for high-level expression of heterologous proteins.
An intravenously injectable liquid formulation of the poorly water-soluble isosteviol sodium (ISVNa) that has a great clinical potential for cardiovascular diseases was developed using the co-solvent technology. The pH and composition of the co-solvent were optimized to obtain a stable liquid formulation (termed as STVNa) based on saline at pH 10.0 containing 25% (v/v) of ethanol and 20% (v/v) of propylene glycol. STVNa was physicochemically stable upon storage for more than 3 months under various conditions. In vitro studies showed that STVNa did not induce hemolytic effects up to 9.1% (v/v) after 3 h of incubation and it was cytocompatible up to 50 μg/mL in H2C9 cells. Furthermore, STVNa showed acceptable safety and pharmacokinetic parameters comparable with those of ISVNa in saline (dissolved at 60 °C) upon i.v. injection in Wistar rats. Overall, the results demonstrated that STVNa is a promising formulation of ISVNa for clinical translation.
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