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

• DOI: 10.1007/s00284-019-01783-9
• Publication Date: 2019-10-14T12:04:19Z
• Authors: Vuong Duong Le; Trang Thi Phuong ...; Tri Minh Nguyen; Luc Brunsveld; Wolfgang Schumann; Hoang Duc Nguyen
• Keywords: Isopropyl Thiogalactoside; Lysine-tRNA Ligase; Cytoplasm; 0303 health sciences; Rhinovirus; Recombinant Fusion Proteins; Green Fluorescent Proteins; 3C Viral Proteases; Gene Expression; beta-Galactosidase; Cysteine Endopeptidases; Industrial Microbiology; Viral Proteins; 03 medical and health sciences; Solubility; Cloning, Molecular; Promoter Regions, Genetic; Bacillus subtilis

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.