Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well.

Cytotoxic Screening of Marine Organisms from Persian Gulf.

BACKGROUND: Marine organisms produce a variety of compounds with pharmacological activities, including anticancer effects. They contain several secondary metabolites with interesting biological activities. This study attempted to find cytotoxicity of Hexane, Dichloromethane and Butanol partitions of Holothuria leucospilota and Echinometra mathaei. MATERIALS AND METHODS: H. leucospilota and E. mathaei were collected from Persian Gulf. The animals were extracted by maceration with methanol-ethyl acetate (1:1). The H. leucospilota extract was partitioned by Kupchan method to hexane, dichloromethane, butanol, and water partitions. The cytotoxic activity of the extracts was investigated against HeLa (cervical cancer) and human umbilical vein endothelial cells cell lines by mitochondrial tetrazolium test assay after 72 h. RESULTS: The cell survivals of HeLa cell were decreased by increasing the concentration of extracts. A significant reduction in cell viability at the doses of 30 (µg/ml) of dichloromethane (DCM) partition, 0.3, 3, and 30 (µg/ml) of ButOH partitions of sea cucumber, and 0.5 (µg/ml) of E. mathaei was observed. The median growth inhibitory concentration value of Hex, DCM, ButoH, and water partitions were 0.301, 0.21, 2.29, and 0.229 µg/ml, respectively. CONCLUSION: This study reveals that different partitions of H. leucospilota and total extract of E. mathaei have cytotoxic activity against cancer cell lines. More study is necessary to find the active metabolites in the more active partitions.