Improvement of Soluble Production of Reteplase in Escherichia Coli by Optimization of Chemical Chaperones in Lysis Buffer

Fazeli, Behnaz; Akbari, Vajihe; Barkhordari, Asgar; Mohammad Sadeghi, Hamid Mir

Improvement of Soluble Production of Reteplase in Escherichia Coli by Optimization of Chemical Chaperones in Lysis Buffer

Document Type : Original Article

Authors

Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Reteplase is a nonglycosylated derivative of recombinant tissue plasminogen activator, a thrombolytic agent, which can be easily expressed in Escherichia coli. However, overexpression of reteplase in E. coli usually leads to accumulation of insoluble and inactive aggregates and inclusion bodies. In the present study, we aimed to optimize chemical additives of lysis buffer to avoid the initial aggregation and formation of inclusion bodies of reteplase at cell disruption step. Materials and Methods: After protein expression in E. coli BL21 (DE3), the bacterial cells were disrupted in different lysis buffers using microsmashing. Eleven chemical additives at two concentration levels were combined based on a Plackett–Burman design to prepare 12 different lysis buffers used at cell disruption stage. Then, three additives with the most positive effect on improvement of solubility of reteplase were chosen and used for the second screening based on Box–Behnken model. Results: The primary screening results showed that among 11 additives, arginine, K₂PO_4,and cetyltrimethylammonium bromide (CTAB) had the most positive effect on solubility of reteplase. Our final results based on 14 runs of Box–Behnken design showed that the optimum buffer additive condition is 0.005 mg/ml CTAB, 0.065 mg/ml arginine, and 0.026 mg/ml K₂PO₄. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis and Western blotting of soluble and total fraction of samples confirmed that these additives significantly improved soluble production of reteplase compared with control. Conclusion: Our study indicates that the application of chemical additives in cell lysis can improve the solubility of reteplase. Further studies are still required to understand the exact mechanism of chemical additives as a chemical chaperone during cell lysis.

Keywords

References

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