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Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli
Journal article   Peer reviewed

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

Hooria Seyedhosseini Ghaheh, Shabnam Sajjadi, Fatemeh Shafiee, Ebrahim Barzegari, Fatemeh Moazen and Hamid Mir Mohammad Sadeghi
World journal of microbiology & biotechnology, Vol.38(2), pp.1-12
01/02/2022
DOI: https://doi.org/10.1007/s11274-021-03204-1
PMID: 34989886

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Abstract

Expression optimization Escherichia coli Molecular dynamic simulation Rational design Reteplase Response surface methodology
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.

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