{"title":"Optimization of expression and renaturation methods for the production of a recombinant fibrinolytic protease showing in vivo antithrombotic activity","authors":"Nitisha Boro , Anushree Roy , Ashis K. Mukherjee","doi":"10.1016/j.pep.2025.106737","DOIUrl":null,"url":null,"abstract":"<div><div>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 <em>Bacillus subtilis</em> was codon-optimized according to <em>Escherichia coli</em> 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 <em>Bacillus</em>. 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 <em>E. coli</em> (DE3) strain. The recombinant protein demonstrated <em>in vitro</em> fibrinolytic, anticoagulant, thrombin-inhibition, and thrombolytic activities but did not show fibrinogenolytic or <em>in vitro</em> 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 <em>in vivo</em> anticoagulant activity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106737"},"PeriodicalIF":1.2000,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592825000798","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
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.
期刊介绍:
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.