Using the IPTG-inducible Pgrac212 promoter for overexpression of human rhinovirus 3C protease fusions in the cytoplasm of bacillus subtilis cells

Vuong Duong Le, Trang Thi Phuong Phan, Tri Minh Nguyen, Luc Brunsveld, Wolfgang Schumann, Hoang Duc Nguyen (Corresponding author)

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Samenvatting

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.

Originele taal-2Engels
Pagina's (van-tot)1477-1486
Aantal pagina's10
TijdschriftCurrent Microbiology
Volume76
Nummer van het tijdschrift12
DOI's
StatusGepubliceerd - 1 dec 2019

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