RESUMO
BACKGROUND: Factor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. METHODS AND RESULTS: The FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. CONCLUSIONS: Protein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.
RESUMO
The present study was intended to prepare and optimize the mucoadhesive buccal patch of gambier leaf extract using chitosan (CH) and tapioca starch (TS) composite as the polymer complexes. The patch formulation was designed based on 22 factorial design in order to optimize the composition of CH and TS. The physical and chemical characteristics of the prepared patches, including mass and thickness uniformity, folding endurance, surface pH, swelling index, percent of elongation, and mucoadhesive time were successfully evaluated. Based on statistical analysis, the optimum concentration of CH and TS was 900 mg and 300 mg, respectively, with desirability percent of 0.968. The characterization of the optimum patch showed that the variability coefficient of the mass and thickness uniformity was 0.4805 ± 0.1887% and 0.9716 ± 1.2026%, surface pH of the patch was 6, folding endurance >300 times, elongation percent was 53.333 ± 0.1082%, and mucoadhesive time was 320 ± 1.1547 min. The catechin content, as the active agent of the gambier leaf extract, was 92.1667 ± 0.3626%, and the FT-IR characterization indicated that there are no chemical interactions between each patch component.
