Investigation of Supercharging as A Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase
Author(s):
Article Type:
Research/Original Article (دارای رتبه معتبر)
Abstract:
Background
Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase.Objective
To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity.Materials and Methods
Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity.Results
SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activityof the supercharged variant than the wild type protein (P < 0.001). The stability of the supercharged variant was also comparable to the wild type.Conclusion
Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.Keywords:
Language:
English
Published:
Iranian Journal of Biotechnology, Volume:18 Issue: 4, Autumn 2020
Pages:
124 to 131
https://magiran.com/p2221178
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