Journal
JOURNAL OF BIOTECHNOLOGY
Volume 231, Issue -, Pages 65-71Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2016.05.034
Keywords
Nattokinase; Prokaryotic expression; Inclusion body; Renaturation
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Funding
- Natural Science Funds of Guangdong Province [2014A030313423]
- Guangdong Provincial Science and Technology program [2014A010107026]
- Specialized Research Fund for the Doctoral Program of Higher Education [20134407120004]
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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 TrisHCI 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. (C) 2016 Elsevier B.V. All rights reserved.
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