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Efficient System of Artificial Oil Bodies for Functional Expression and Purification of Recombinant Nattokinase in Escherichia coli

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Department of Life Science and Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, and Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan
Cite this: J. Agric. Food Chem. 2005, 53, 12, 4799–4804
Publication Date (Web):May 3, 2005
https://doi.org/10.1021/jf050264a
Copyright © 2005 American Chemical Society
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    Abstract

    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin−intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin−intein−pronattokinase exhibited 5 times higher activity than that released from oleosin−intein−nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

    Keywords: Artificial oil body; intein; nattokinase; oleosin; propeptide

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    †

     Department of Life Science, National Chung-Hsing University.

    ‡

     Feng Chia University.

    *

     Author to whom correspondence should be addressed (telephone 886-4-22840328; fax 886-4-22853527; e-mail [email protected]).

    §

     Graduate Institute of Biotechnology, Natinal Chung-Hsing University.

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    8. Daigo Iwanaga, David A. Gray, Ian D. Fisk, Eric Andrew Decker, Jochen Weiss and David Julian McClements. Extraction and Characterization of Oil Bodies from Soy Beans: A Natural Source of Pre-Emulsified Soybean Oil. Journal of Agricultural and Food Chemistry 2007, 55 (21) , 8711-8716. https://doi.org/10.1021/jf071008w
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    14. Yuan Li, Xiyu Tang, Liangqi Chen, Xinran Xu, Jinyao Li. Characterization of a Nattokinase from the Newly Isolated Bile Salt-Resistant Bacillus mojavensis LY-06. Foods 2022, 11 (16) , 2403. https://doi.org/10.3390/foods11162403
    15. Jia Hao, Xiaoyu Li, Qiuyu Wang, Wenwen Lv, Wenguan Zhang, Duoxia Xu. Recent developments and prospects in the extraction, composition, stability, food applications, and in vitro digestion of plant oil bodies. Journal of the American Oil Chemists' Society 2022, 99 (8) , 635-653. https://doi.org/10.1002/aocs.12618
    16. Li Yuan, Chen Liangqi, Tang Xiyu, Li Jinyao. Biotechnology, Bioengineering and Applications of Bacillus Nattokinase. Biomolecules 2022, 12 (7) , 980. https://doi.org/10.3390/biom12070980
    17. Yan Guangbo, Shu Min, Shen Wei, Ma Lixin, Zhai Chao, Wang Yaping, Huang Zunxi. Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity. BMC Biotechnology 2021, 21 (1) https://doi.org/10.1186/s12896-021-00708-4
    18. Chhavi Sharma, Alexander Osmolovskiy, Rajni Singh. Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications. Pharmaceutics 2021, 13 (11) , 1880. https://doi.org/10.3390/pharmaceutics13111880
    19. Jason T. C. Tzen. Beneficial Components in Sesame Proteins and Oil. 2021, 59-78. https://doi.org/10.1007/978-3-319-98098-0_3
    20. Zitong Zhao, Fusheng Chen, Lihua Hao. Effect of Physiochemical Factors and Peanut Varieties on the Charge Stability of Oil Bodies Extracted by Aqueous Method. Journal of Oleo Science 2019, 68 (4) , 297-306. https://doi.org/10.5650/jos.ess18157
    21. Farah Zaaboul, Husnain Raza, Abderrahim Lazraq, Boxin Deng, Chen Cao, Yuan F. Liu. Chemical Composition, Physical Properties, and the Oxidative Stability of Oil Bodies Extracted From Argania spinosa. Journal of the American Oil Chemists' Society 2018, 95 (4) , 485-495. https://doi.org/10.1002/aocs.12053
    22. Essam Kotb. Microbial Fibrinolytic Enzyme Production and Applications. 2017, 175-213. https://doi.org/10.1002/9781119048961.ch8
    23. Dongbo Cai, Chengjun Zhu, Shouwen Chen. Microbial production of nattokinase: current progress, challenge and prospect. World Journal of Microbiology and Biotechnology 2017, 33 (5) https://doi.org/10.1007/s11274-017-2253-2
    24. Yunqi Weng, Jian Yao, Sawyer Sparks, Kevin Wang. Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease. International Journal of Molecular Sciences 2017, 18 (3) , 523. https://doi.org/10.3390/ijms18030523
    25. Longjun Dai, Zhiyi Nie, Guijuan Kang, Yu Li, Rizhong Zeng. Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles. Plant Physiology and Biochemistry 2017, 111 , 97-106. https://doi.org/10.1016/j.plaphy.2016.11.006
    26. Ruifeng Mao, Kangping Zhou, Zhenwei Han, Yefu Wang. Subtilisin QK-2: secretory expression in Lactococcus lactis and surface display onto gram-positive enhancer matrix (GEM) particles. Microbial Cell Factories 2016, 15 (1) https://doi.org/10.1186/s12934-016-0478-7
    27. Katarina Bílikova, Sheng-Chang Huang, I-Ping Lin, Jozef Šimuth, Chi-Chung Peng. Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera. Peptides 2015, 68 , 190-196. https://doi.org/10.1016/j.peptides.2015.03.001
    28. Natalie Laibach, Janina Post, Richard M. Twyman, Christian Schulze Gronover, Dirk Prüfer. The characteristics and potential applications of structural lipid droplet proteins in plants. Journal of Biotechnology 2015, 201 , 15-27. https://doi.org/10.1016/j.jbiotec.2014.08.020
    29. Lan Han, Liquan Zhang, Jinglei Liu, Hong Li, Yingchun Wang, Agula Hasi. Transient expression of optimized and synthesized nattokinase gene in melon (Cucumis melo L.) fruit by agroinfiltration. Plant Biotechnology 2015, 32 (2) , 175-180. https://doi.org/10.5511/plantbiotechnology.15.0430a
    30. Fatemeh Dabbagh, Manica Negahdaripour, Aydin Berenjian, Abdolazim Behfar, Fatemeh Mohammadi, Mozhdeh Zamani, Cambyz Irajie, Younes Ghasemi. Nattokinase: production and application. Applied Microbiology and Biotechnology 2014, 98 (22) , 9199-9206. https://doi.org/10.1007/s00253-014-6135-3
    31. Fangwen Bai, Wei Yan, Shujun Zhang, Dong Yu, Linhan Bai. Immobilized lipase of reconstructed oil bodies and its potential application in biodiesel production. Fuel 2014, 128 , 340-346. https://doi.org/10.1016/j.fuel.2014.03.033
    32. Essam Kotb. The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi. Biotechnology Progress 2014, 30 (3) , 656-672. https://doi.org/10.1002/btpr.1918
    33. Ana Sofia Pina, Christopher R. Lowe, Ana Cecília A. Roque. Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnology Advances 2014, 32 (2) , 366-381. https://doi.org/10.1016/j.biotechadv.2013.12.001
    34. Chen Chen, Haogang Duan, Chunmei Gao, Mingzhu Liu, Xin'an Wu, Yuhui Wei, Xinyu Zhang, Zhen Liu. Non-covalent modification of thrombolytic agent nattokinase: simultaneous improvement of fibrinolysis activity and enzymatic stability. RSC Adv. 2014, 4 (52) , 27422-27429. https://doi.org/10.1039/C4RA02626H
    35. Chung-Jen Chiang, Po Ting Chen, Chien Yu Yeh, Yun-Peng Chao. Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum. Process Biochemistry 2013, 48 (12) , 1886-1892. https://doi.org/10.1016/j.procbio.2013.08.022
    36. Chung-Jen Chiang, Po Ting Chen, Chien Yu Yeh, Zei Wen Wang, Yun-Peng Chao. A useful method integrating production and immobilization of recombinant cellulase. Applied Microbiology and Biotechnology 2013, 97 (20) , 9185-9192. https://doi.org/10.1007/s00253-013-5238-6
    37. Essam Kotb. Activity assessment of microbial fibrinolytic enzymes. Applied Microbiology and Biotechnology 2013, 97 (15) , 6647-6665. https://doi.org/10.1007/s00253-013-5052-1
    38. Samuel Furse, Susan Liddell, Catharine A. Ortori, Huw Williams, D. Cameron Neylon, David J. Scott, David A. Barrett, David A. Gray. The lipidome and proteome of oil bodies from Helianthus annuus (common sunflower). Journal of Chemical Biology 2013, 6 (2) , 63-76. https://doi.org/10.1007/s12154-012-0090-1
    39. Ming‐Tsung Chang, Chun‐Ren Chen, Ting‐Hang Liu, Chin‐Pei Lee, Jason TC Tzen. Development of a protocol to solidify native and artificial oil bodies for long‐term storage at room temperature. Journal of the Science of Food and Agriculture 2013, 93 (6) , 1516-1519. https://doi.org/10.1002/jsfa.5870
    40. Jason T. C. Tzen. Integral Proteins in Plant Oil Bodies. ISRN Botany 2012, 2012 , 1-16. https://doi.org/10.5402/2012/173954
    41. Chung-Jen Chiang, Shen-Chuan Lin, Li-Jen Lin, Chih-Jung Chen, Yun-Peng Chao. Caleosin-assembled oil bodies as a potential delivery nanocarrier. Applied Microbiology and Biotechnology 2012, 93 (5) , 1905-1915. https://doi.org/10.1007/s00253-011-3716-2
    42. Chi-Cung Peng, Jia-Yang Hong, Wu-Chun Tu. Cost-Effective Expression and Purification of Recombinant Venom Peptide Mastoparan B of Vespa Basalis in Escherichia Coli. International Journal of Bioscience, Biochemistry and Bioinformatics 2012, , 309-313. https://doi.org/10.7763/IJBBB.2012.V2.123
    43. Essam Kotb. Fibrinolytic Bacterial Enzymes with Thrombolytic Activity. 2012, 1-74. https://doi.org/10.1007/978-3-642-24980-8_1
    44. Ekkarat Phrommao, Jirawat Yongsawatdigul, Sureelak Rodtong, Montarop Yamabhai. A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37. BMC Biotechnology 2011, 11 (1) https://doi.org/10.1186/1472-6750-11-65
    45. Ting‐Hang Liu, Chia‐Lin Chyan, Feng‐Yin Li, Ying‐Jie Chen, Jason T. C. Tzen. Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production. Biotechnology Progress 2011, 27 (6) , 1760-1767. https://doi.org/10.1002/btpr.684
    46. Chung-Jen Chiang, Che-Chin Lin, Tzu-Li Lu, Hesin-Fu Wang. Functionalized nanoscale oil bodies for targeted delivery of a hydrophobic drug. Nanotechnology 2011, 22 (41) , 415102. https://doi.org/10.1088/0957-4484/22/41/415102
    47. Cerrone Cabanos, Hiroki Katayama, Akira Tanaka, Shigeru Utsumi, Nobuyuki Maruyama. Expression and Purification of Peanut Oleosins in Insect Cells. The Protein Journal 2011, 30 (7) , 457-463. https://doi.org/10.1007/s10930-011-9351-z
    48. Yifeng Li. Self-cleaving fusion tags for recombinant protein production. Biotechnology Letters 2011, 33 (5) , 869-881. https://doi.org/10.1007/s10529-011-0533-8
    49. Jiun-Yan Wu, Tsung-Yu Tsai, Tzu-Tsen Liu, Chia-Chi Lin, Jiann-Hwa Chen, Shih-Chun Yang, Chwen-Jen Shieh, Yung-Chuan Liu. Production of recombinant EGFP via surface display of ice nucleation protein and self-cleavage intein. Biochemical Engineering Journal 2011, 54 (3) , 158-163. https://doi.org/10.1016/j.bej.2011.02.009
    50. Chung-Jen Chiang, Li-Jen Lin, Che-Chin Lin, Chih-Hsiang Chang, Yun-Peng Chao. Selective internalization of self-assembled artificial oil bodies by HER2 /neu -positive cells. Nanotechnology 2011, 22 (1) , 015102. https://doi.org/10.1088/0957-4484/22/1/015102
    51. Jun‐Ming Tseng, Jun‐Ru Huang, Hsiou‐Chen Huang, Jason T. C. Tzen, Wing‐Ming Chou, Chi‐Chung Peng. Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil‐body system. Biotechnology Progress 2011, 27 (1) , 153-161. https://doi.org/10.1002/btpr.528
    52. Richard W. Scott, Somrutai Winichayakul, Marissa Roldan, Ruth Cookson, Melanie Willingham, Maria Castle, Ringo Pueschel, Chi-Chung Peng, Jason T. C. Tzen, Nicholas J. Roberts. Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head-to-tail fusions. Plant Biotechnology Journal 2010, 8 (8) , 912-927. https://doi.org/10.1111/j.1467-7652.2010.00522.x
    53. S.C. Bhatla, V. Kaushik, M.K. Yadav. Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications. Biotechnology Advances 2010, 28 (3) , 293-300. https://doi.org/10.1016/j.biotechadv.2010.01.001
    54. Yi‐Fang Zeng, Ying‐Jing Hung, Ming‐Ju Chen, Chi‐Chung Peng, Jason T. C. Tzen, Je‐Ruei Liu. Simultaneous refolding, purification, and immobilization of recombinant Fibrobacter succinogenes 1,3‐1,4‐β‐D‐glucanase on artificial oil bodies. Journal of Chemical Technology & Biotechnology 2009, 84 (10) , 1480-1485. https://doi.org/10.1002/jctb.2205
    55. Seung Phill Choi, Ho Nam Chang. Stable constitution of artificial oil body for the refolding of IGF1. Biotechnology and Bioprocess Engineering 2009, 14 (2) , 161-167. https://doi.org/10.1007/s12257-008-0157-6
    56. Ying-Jing Hung, Chi-Chung Peng, Jason T.C. Tzen, Ming-Ju Chen, Je-Ruei Liu. Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity. Bioresource Technology 2008, 99 (18) , 8662-8666. https://doi.org/10.1016/j.biortech.2008.04.017
    57. Je-Ruei Liu, Chung-Hang Duan, Xin Zhao, Jason T. C. Tzen, Kuo-Joan Cheng, Cheng-Kang Pai. Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies. Applied Microbiology and Biotechnology 2008, 79 (2) , 225-233. https://doi.org/10.1007/s00253-008-1418-1
    58. Kyoung-Ji Chung, Seon-Kap Hwang, Bum-Soo Hahn, Kyung-Hwan Kim, Jong-Bum Kim, Yong-Hwan Kim, Joo-Sung Yang, Sun-Hwa Ha. Authentic seed-specific activity of the Perilla oleosin 19 gene promoter in transgenic Arabidopsis. Plant Cell Reports 2007, 27 (1) , 29-37. https://doi.org/10.1007/s00299-007-0440-6
    59. Xiaobo Liang, Shifang Jia, Yufang Sun, Meiling Chen, Xiuzhu Chen, Jin Zhong, Liandong Huan. Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli. Molecular Biotechnology 2007, 37 (3) , 187-194. https://doi.org/10.1007/s12033-007-0060-y
    60. Hyun Kim, So-Young Kim, Nam Soo Han, Bernard Y. Tao. Solubilization conditions for hydrophobic membrane protein, oleosin, in soybeans. Biotechnology and Bioprocess Engineering 2007, 12 (5) , 542-547. https://doi.org/10.1007/BF02931352
    61. Xiaobo Liang, Lixin Zhang, Jin Zhong, Liandong Huan. Secretory expression of a heterologous nattokinase in Lactococcus lactis. Applied Microbiology and Biotechnology 2007, 75 (1) , 95-101. https://doi.org/10.1007/s00253-006-0809-4
    62. Hua Ling. Oleosin fusion expression systems for the production of recombinant proteins. Biologia 2007, 62 (2) , 119-123. https://doi.org/10.2478/s11756-007-0041-4
    63. Chung-Jen Chiang, Hong-Chen Chen, Yun-Peng Chao, Jason T.C. Tzen. One-step purification of insoluble hydantoinase overproduced in Escherichia coli. Protein Expression and Purification 2007, 52 (1) , 14-18. https://doi.org/10.1016/j.pep.2006.07.008
    64. Chung-Jen Chiang, Hong-Chen Chen, Hsueh-Feng Kuo, Yun-Peng Chao, Jason T.C. Tzen. A simple and effective method to prepare immobilized enzymes using artificial oil bodies. Enzyme and Microbial Technology 2006, 39 (5) , 1152-1158. https://doi.org/10.1016/j.enzmictec.2006.02.024
    65. Yong Peng, Xiaojuan Yang, Yizheng Zhang. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Applied Microbiology and Biotechnology 2005, 69 (2) , 126-132. https://doi.org/10.1007/s00253-005-0159-7
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