High yield expression and purification of Aspergillus flavus uricase cloned in Pichia pink™ expression system.

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7 U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60 h, the fermentation process produced 1500 g of biomass from 4 L of semi defined culture media and expressed 2.5 g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24 U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study.

Co-Delivery of erlotinib and resveratrol via nanostructured lipid Carriers: A synergistically promising approach for cell proliferation prevention and ROS-Mediated apoptosis activation.

Cancer treatments are always associated with various challenges, and scientists are constantly trying to find new therapies and methods. Erlotinib (ELT) is a well-known medicine against non-small cell lung cancer (NSCLC). However, treatments by ELT disrupt therapy due to drug resistance and pose severe challenges to patients. To achieve high-performance treatment, we gained nanostructured lipid carriers (NLCs) to evaluate synergistic anticancer effects of co-delivery of ELT and resveratrol (RES), a natural herbal derived phenol against NSCLC. NLCs are prepared via the hot homogenization method and characterized. In vitro cytotoxicity of formulations were evaluated on adenocarcinoma human alveolar basal epithelial (A549) cells. Prepared NLCs showed a narrow particle size (97.52 ± 17.14 nm), negative zeta potential (-7.67 ± 4.55 mV), and high encapsulation efficiency (EE%) was measured for the prepared co-delivery system (EE% 89.5 ± 5.16 % for ELT and 90.1 ± 6.61 % for RES). In vitro outcomes from cell viability study (12.63 % after 48 h of treatment), apoptosis assay (85.50%.), cell cycle (40.00% arrest in G2-M), and western blotting investigations (decreasing of protein expression levels of survivin, Bcl-2, P-Caspase 3P-caspase 9, and P-ERK 1/2, and additionally, increasing protein levels of BAX, P53, C-Caspase 3 and 9), DAPI staining, and colony formation assays showed the augment cytotoxic performances for co-delivery of ELT and RES loaded NLCs. Our study introduced the co-delivery of ELT and RES by NLCs as a novel strategy to elevate the efficacy of chemotherapeutics for NSCLC.