Optimization of expression and renaturation methods for the production of a recombinant fibrinolytic protease showing in vivo antithrombotic activity.

Bacterial fibrinolytic enzymes are promising in treating thrombosis-associated cardiovascular disease. The recombinant fibrinolytic enzymes exhibiting enhanced specificity and improved pharmacokinetics, being less immunogenic and easy to produce, can be advantageous over wild-type enzymes. However, efficient expression and refolding of recombinant enzymes is a significant challenge; therefore, three different analytical methods were compared in this study for the efficient expression and refolding of a recombinant fibrinolytic protease. The gene sequence encoding for fibrinolytic enzyme derived from Bacillus subtilis was codon-optimized according to Escherichia coli codon preference, and this gene was synthetically cloned into the pET26b(+) vector. Alignment of amino acid sequence of this protease gene revealed high sequence similarity with other species of the genus Bacillus. 24 h induced expression at 37°C and dialysis for renaturation was the most suitable process for expression (enzyme yield) and refolding or renaturation of a ∼40 kDa recombinant α-fibrinogenase enzyme produced in the E. coli (DE3) strain. The recombinant protein demonstrated in vitro fibrinolytic, anticoagulant, thrombin-inhibition, and thrombolytic activities but did not show fibrinogenolytic or in vitro cytotoxicity activity. At a dose of 4 mg/kg, it was found to be non-toxic to Wistar strain albino rats post 72 h of injection but demonstrated dose-dependent in vivo anticoagulant activity.

• Publication year

  2025

• Venue

  Protein Expression and Purification

• Publication date

  2025-05-01

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.pep.2025.106737PMID 40374132
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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