Auto-induction for high level production of biologically active reteplase in Escherichia coli
| Autor: | Morvarid Soleiman, Mehrnoosh Fathi-Roudsari, Sepideh Niazi, Nader Maghsoudi, Amirhossein Maghsoudi |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Cytoplasm Protein Folding Protein Conformation Reteplase medicine.disease_cause 01 natural sciences Tissue plasminogen activator law.invention 03 medical and health sciences Protein structure law 010608 biotechnology medicine Escherichia coli Chemistry Biological activity Recombinant Proteins 030104 developmental biology Biochemistry Gene Expression Regulation Tissue Plasminogen Activator Recombinant DNA Protein folding Biotechnology medicine.drug |
| Zdroj: | Protein expression and purification. 151 |
| ISSN: | 1096-0279 |
| Popis: | Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E. coli. Results showed that using auto-induction strategy at 37 °C, Rosetta-gami (DE3) had the highest level of active and soluble reteplase production in comparison to E. coli strains BL21 (DE3), and Shuffel T7. Temperature dominantly affected the level of active reteplase production. Decreasing the temperature to 25 and 18 °C increased the level of active reteplase by 20 and 60%, respectively. The composition of auto-induction medium also dramatically changed the active production of reteplase in cytoplasm. Using higher enriched auto-induction medium, super broth base including trace elements, significantly increased biologically active reteplase by 30%. It is demonstrated here that auto-induction is a powerful method for expression of biologically active reteplase in oxidative cytoplasm of Rosetta-gami. Optimizing expression condition by decreasing temperature and using an enriched auto-induction medium resulted in at least three times higher level of active reteplase production. Production of correctly folded and active reteplase in spite of its complex structure helps for removal of inefficient and cumbersome step of refolding. |
| Databáze: | OpenAIRE |
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