In Silico Modeling and Molecular Cloning of Reteplase in A Prokaryotic System

Introduction: In 2024, cardiovascular diseases (CVDs) remained the leading cause of death globally, accounting for approximately 17.9 million deaths. The majority of these fatalities — around 85% — were due to heart attacks and strokes. Objective: The objective of this study was to produce reteplase in vitro for its clot lysis activity and to investigate the reteplase–plasminogen thermodynamically. Methodology: The Modeller software was used for protein modeling. Plasminogen was docked with the reteplase protein using ClusPro software. Desmond and GROMACS software were used for MD simulation to check the stability of the protein complex thermodynamically. In experimental assessment, the reteplase gene was amplified and primarily cloned into the pTZ57R/T vector. The gene was further cloned into expression vectors pET28a ([Formula: see text] and pET22b ([Formula: see text]. The cloned gene was expressed in BL21-competent cells for protein expression. A clot lysis assay of crude protein was performed. Results: The model with the lowest dope score was selected and docked with plasminogen. The docked complex was used for protein MD simulation, which showed that the complex was thermodynamically stable. Experimentally, the reteplase gene was cloned and expressed in BL21 cells. The protein expression was observed at 39 kDa on SDS-PAGE. A 300 [Formula: see text]L crude reteplase sample showed significant clot lysis activity, whereas the 400[Formula: see text]L and 500 [Formula: see text]L samples showed decreased activity.