Abstract
Purpose: Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized in the treatment of vitreomacular adhesion (VMA). This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any Oor 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 highlevel 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 was greater than that of 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.
Highlights
Ocriplasmin, known as microplasmin, represents a novel pharmacologic agent approved by the Food and Drug Administration
The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403
We evaluated E. coli expression system for the production of active ocriplasmin
Summary
Ocriplasmin, known as microplasmin, represents a novel pharmacologic agent approved by the Food and Drug Administration It is a recombinant truncated human plasmin which is used for the treatment of vitreoretinopathies including symptomatic VMA.[1,2,3] Abnormal symptomatic VMA is an age-related disorder that occurred due to partial PVD. This can exert traction on the retina and may result in visual distortions and even macular holes that in more serious cases leads to loss of vision.[4] Till the FDA approval of ocriplasmin, the only treatment for VMA was a surgical method as vitrectomy. This method has the risk of retinal damage,[5,6] that describes why some of proteolytic enzymes, containing plasmin, have been experienced for enzymatic release of retinal traction.[7,8,9,10] Ocriplasmin is a nonsurgical treatment option to induce posterior vitreous detachment due to its potential for degradation of fibronectin and laminin after intravitreal injection.[11,12,13,14]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
