title: Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.
abstract: 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 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM 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 42.8IU/mg and 79.3IU/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 52.0IU/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.
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China.
authors.forename: He
authors.initials: H
authors.lastname: Ni
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China.
authors.forename: Peng-Cheng
authors.initials: PC
authors.lastname: Guo
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China.
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Jiang
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Fan
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China; Guangzhou Huichuan Medical Technology Ltd., 211 Jinfu Building, 90 Qifu Road, Baiyun District, Guangzhou 510410, China.
authors.forename: Xiang-Yu
authors.initials: XY
authors.lastname: Luo
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal University, Guangzhou 510631, China. Electronic address: li_haihang@yahoo.com.
authors.forename: Hai-Hang
authors.initials: HH
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D058849:Protein Refolding; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005343:Fibrinolytic Agents; D013381:Subtilisins; C053771:nattokinase
keywords: Inclusion body; Nattokinase; Prokaryotic expression; Renaturation