Rational Design of a New Variant of Reteplase With Optimized Physicochemical Profile and Large-Scale Production in Escherichia Coli Publisher Pubmed

Summary: Could a new Reteplase variant improve thrombolytic therapy? Research suggests M72R shows better stability and activity, boosting production levels. How could this affect treatment? #Pharmaceuticals #Biotechnology

Seyedhosseini Ghaheh H¹ ; Sajjadi S² ; Shafiee F¹ ; Barzegari E³ ; Moazen F¹ ; Mir Mohammad Sadeghi H¹
Source: World Journal of Microbiology and Biotechnology Published:2022

Abstract

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. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.