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  Abstract View: 68

Research Article

Assessment of E. coli expression system for overexpression of active recombinant ocriplasmin

Roghayyeh Baghban ORCID logo, Safar Farajnia* ORCID logo, Younes Ghasemi, Mojtaba Mortazavi, Naser Samadi, Nosratollah Zarghami

Abstract

Purpose: Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized to treat a human medical condition called vitreomacular adhesion (VMA). The safety and effectiveness of ocriplasmin for VMA treatment has been confirmed in clinical studies. This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any O- or N-glycosylation or some other post-translational modifications, bacterial expression systems such as Escherichia coli could be considered as an economical host for recombinant expression. In the present study, we aimed to evaluate the efficiency of E. coli expression system for high-level expression of recombinant ocriplasmin. Methods: The gene coding for ocriplasmin was cloned and expressed in E. coli BL21. The bacterial cells were cultured on large scale and the expressed recombinant protein was purified using Ni-NTA chromatography. Refolding of denatured ocriplasmin to active enzyme was carried out by the stepwise removal of denaturant. The identity of recombinant ocriplasmin was confirmed using western blotting and ELISA assays. The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403. Results: The final yield of E. coli BL21-produced ocriplasmin was approximately 1 mg/ml which is greater than that produced by P. pastoris. Using western blotting and ELISA assay, the identity of recombinant ocriplasmin was confirmed. The hydrolysis of chromogenic substrate S-2403 verified the functional activity of E. coli produced ocriplasmin. Conclusion: The results of this study indicated that E. coli could be used for high level expression of ocriplasmin. Although the recombinant protein was expressed as inclusion body, the stepwise refolding leads to the biologically active proteins that can potentially be used for therapeutic purposes.
Keywords: Ocriplasmin, Recombinant expression, Vitreomacular adhesion (VMA)
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Submitted: 22 Jan 2020
Revision: 24 Apr 2020
Accepted: 21 Jun 2020
ePublished: 21 Jun 2020
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