Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity
Background: Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Objectives: Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity. Meth...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-05-01
|
| Series: | Arabian Journal of Chemistry |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1878535222001150 |
| _version_ | 1811279477455454208 |
|---|---|
| author | Ehab El-Dabaa Hend Okasha Safia Samir Sami Mohamed Nasr Hadeer Adel El-Kalamawy Mohamed Ali Saber |
| author_facet | Ehab El-Dabaa Hend Okasha Safia Samir Sami Mohamed Nasr Hadeer Adel El-Kalamawy Mohamed Ali Saber |
| author_sort | Ehab El-Dabaa |
| collection | DOAJ |
| description | Background: Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Objectives: Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity. Methods: Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Results: Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x106 IU/mg with 62.94 ± 2.3% clot lysis efficiency. Conclusion: A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug. |
| first_indexed | 2024-04-13T00:55:18Z |
| format | Article |
| id | doaj.art-6f0152ff86b7418f8afc005c3562b4bf |
| institution | Directory Open Access Journal |
| issn | 1878-5352 |
| language | English |
| last_indexed | 2024-04-13T00:55:18Z |
| publishDate | 2022-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Arabian Journal of Chemistry |
| spelling | doaj.art-6f0152ff86b7418f8afc005c3562b4bf2022-12-22T03:09:42ZengElsevierArabian Journal of Chemistry1878-53522022-05-01155103799Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activityEhab El-Dabaa0Hend Okasha1Safia Samir2Sami Mohamed Nasr3Hadeer Adel El-Kalamawy4Mohamed Ali Saber5Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptDepartment of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptDepartment of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptCorresponding author at: Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, Egypt.; Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptDepartment of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptDepartment of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza 12411, EgyptBackground: Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Objectives: Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity. Methods: Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Results: Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x106 IU/mg with 62.94 ± 2.3% clot lysis efficiency. Conclusion: A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.http://www.sciencedirect.com/science/article/pii/S1878535222001150StreptokinaseExpressionRefoldingPurificationActivity assays |
| spellingShingle | Ehab El-Dabaa Hend Okasha Safia Samir Sami Mohamed Nasr Hadeer Adel El-Kalamawy Mohamed Ali Saber Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity Arabian Journal of Chemistry Streptokinase Expression Refolding Purification Activity assays |
| title | Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| title_full | Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| title_fullStr | Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| title_full_unstemmed | Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| title_short | Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| title_sort | optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity |
| topic | Streptokinase Expression Refolding Purification Activity assays |
| url | http://www.sciencedirect.com/science/article/pii/S1878535222001150 |
| work_keys_str_mv | AT ehabeldabaa optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity AT hendokasha optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity AT safiasamir optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity AT samimohamednasr optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity AT hadeeradelelkalamawy optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity AT mohamedalisaber optimizationofhighexpressionandpurificationofrecombinantstreptokinaseandinvitroevaluationofitsthrombolyticactivity |