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Expression of nattokinase in Escherichia coli and renaturation of its inclusion body

Paper ID Volume ID Publish Year Pages File Format Full-Text
22667 43369 2016 7 PDF Available
Title
Expression of nattokinase in Escherichia coli and renaturation of its inclusion body
Abstract

•The mature nattokinase gene expressed in E. coli existed as inclusion bodies without enzyme activity.•The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10.•The refolded proteins showed 79.3 IU/mg fibrinolytic activity by the directly mixing the protein solution with equal volume RS-10.

Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1 mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20 mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8 IU/mg and 79.3 IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0 IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution.

Keywords
Nattokinase; Prokaryotic expression; Inclusion body; Renaturation
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Expression of nattokinase in Escherichia coli and renaturation of its inclusion body
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 231, 10 August 2016, Pages 65–71
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us