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Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells

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Abstract

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni–NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.

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Funding

This research was funded by the Department of Science and Technology of Ho Chi Minh City, Vietnam under Grant Number 1022/QĐ-SKHCN.

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Authors and Affiliations

  1. Center for Bioscience and Biotechnology, University of Science-VNUHCM, 227 Nguyen Van Cu Dist. 5, Hochiminh, Vietnam

    Vuong Duong Le, Trang Thi Phuong Phan, Tri Minh Nguyen, Wolfgang Schumann & Hoang Duc Nguyen

  2. Ho Chi Minh City University of Technology (HUTECH), 475A Dien Bien Phu Str., Binh Thanh Dist., Hochiminh, Vietnam

    Vuong Duong Le & Tri Minh Nguyen

  3. Laboratory of Molecular Biotechnology, University of Science-VNUHCM, 227 Nguyen Van Cu Dist. 5, Hochiminh, Vietnam

    Trang Thi Phuong Phan

  4. Laboratory of Chemical Biology & Institute of Complex Molecular Systems, Department of Biomedical Engineering, Technische Universiteit Eindhoven, Eindhoven, Netherlands

    Luc Brunsveld

  5. Institute of Genetics, University of Bayreuth, 95440, Bayreuth, Germany

    Wolfgang Schumann

  6. Department of Microbiology, University of Science-VNUHCM, 227 Nguyen Van Cu Dist. 5, Hochiminh, Vietnam

    Vuong Duong Le & Hoang Duc Nguyen

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  1. Vuong Duong Le
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Correspondence to Hoang Duc Nguyen.

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Le, V.D., Phan, T.T.P., Nguyen, T.M. et al. Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells. Curr Microbiol 76, 1477–1486 (2019). https://doi.org/10.1007/s00284-019-01783-9

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