cloning and expression of serratiopeptidase protein and assessment of its activity for clot lysis

Objectivecardiovascular diseases (cvds) remain a leading cause of morbidity and mortality worldwide, with blood thrombosis playing a critical role in conditions such as myocardial infarction and stroke. thrombolytic agents, including tissue plasminogen activators and streptokinase, are commonly used to dissolve blood clots and restore normal blood flow. however, these agents have several limitations, including high cost, short half-life, immune responses, and potential hemorrhagic complications, which restrict their widespread clinical use. serratiopeptidase, a zinc-dependent metalloprotease produced by serratia marcescens, has gained attention for its fibrinolytic, anti-inflammatory, and analgesic properties. this enzyme has demonstrated effective fibrin clot degradation with minimal side effects, making it a promising candidate for thrombolytic therapy. additionally, its ability to bind to alpha-2-macroglobulin may help mask its antigenicity, potentially reducing immune-related adverse reactions. despite these advantages, large-scale recombinant production of serratiopeptidase is necessary to enhance its availability and therapeutic applications. in this study, we aimed to clone and express the serratiopeptidase (stp) gene in escherichia coli bl21(de3) and evaluate the thrombolytic activity of the recombinant protein. using molecular cloning techniques, followed by protein expression, purification, and fibrinolytic assays, we assessed the efficacy of recombinant serratiopeptidase in clot degradation.materials and methodsthe stp gene was isolated from a pathogenic serratia marcescens strain. the gene was digested with ecor1, ligated into the expression vector pgem®-3zf, and transformed into e. coli bl21(de3). expression was induced using isopropyl β-d-1-thiogalactopyranoside (iptg). the recombinant stp protein was analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). the fibrinolytic activity of the expressed serratiopeptidase protein was assessed using blood clot lysis assays.resultsthe pcr product of the stp gene, approximately 1500 bp in size, was confirmed via agarose gel electrophoresis and sequencing. successful cloning in e. coli was verified using 12% sds-page, which showed a protein band corresponding to the expected molecular weight of 52 kda. functional analysis demonstrated that the recombinant serratiopeptidase effectively lysed human blood clots, showing complete clot degradation compared to the control.conclusionour findings indicate that recombinant serratiopeptidase exhibits significant thrombolytic activity, effectively dissolving human blood clots. these results suggest that serratiopeptidase could serve as a promising alternative to existing thrombolytic agents. further studies are needed to optimize its production, assess its stability and safety, and explore its clinical applications for cardiovascular disease management.

cloning and expression of serratiopeptidase protein and assessment of its activity for clot lysis

objectivecardiovascular diseases (cvds) remain a leading cause of morbidity and mortality worldwide, with blood thrombosis playing a critical role in conditions such as myocardial infarction and stroke. thrombolytic agents, including tissue plasminogen activators and streptokinase, are commonly used to dissolve blood clots and restore normal blood flow. however, these agents have several limitations, including high cost, short half-life, immune responses, and potential hemorrhagic complications, which restrict their widespread clinical use. serratiopeptidase, a zinc-dependent metalloprotease produced by serratia marcescens, has gained attention for its fibrinolytic, anti-inflammatory, and analgesic properties. this enzyme has demonstrated effective fibrin clot degradation with minimal side effects, making it a promising candidate for thrombolytic therapy. additionally, its ability to bind to alpha-2-macroglobulin may help mask its antigenicity, potentially reducing immune-related adverse reactions. despite these advantages, large-scale recombinant production of serratiopeptidase is necessary to enhance its availability and therapeutic applications. in this study, we aimed to clone and express the serratiopeptidase (stp) gene in escherichia coli bl21(de3) and evaluate the thrombolytic activity of the recombinant protein. using molecular cloning techniques, followed by protein expression, purification, and fibrinolytic assays, we assessed the efficacy of recombinant serratiopeptidase in clot degradation.materials and methodsthe stp gene was isolated from a pathogenic serratia marcescens strain. the gene was digested with ecor1, ligated into the expression vector pgem®-3zf, and transformed into e. coli bl21(de3). expression was induced using isopropyl β-d-1-thiogalactopyranoside (iptg). the recombinant stp protein was analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). the fibrinolytic activity of the expressed serratiopeptidase protein was assessed using blood clot lysis assays.resultsthe pcr product of the stp gene, approximately 1500 bp in size, was confirmed via agarose gel electrophoresis and sequencing. successful cloning in e. coli was verified using 12% sds-page, which showed a protein band corresponding to the expected molecular weight of 52 kda. functional analysis demonstrated that the recombinant serratiopeptidase effectively lysed human blood clots, showing complete clot degradation compared to the control.conclusionour findings indicate that recombinant serratiopeptidase exhibits significant thrombolytic activity, effectively dissolving human blood clots. these results suggest that serratiopeptidase could serve as a promising alternative to existing thrombolytic agents. further studies are needed to optimize its production, assess its stability and safety, and explore its clinical applications for cardiovascular disease management.