Abstract
The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The β-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.
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Funding
This work was partially funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106-NN.02–2015.24. The funding agencies had no role in the design, execution, or interpretation of this work. Phuong Thi Bich Chu was funded by Vingroup Joint Stock Company and supported by the Domestic PhD Scholarship Programme of Vingroup Innovation Foundation (VINIF), Vingroup Big Data Institute (VINBIGDATA), code VINIF.2020.TS.29.
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HDN, TTPP and WS conceived the research and designed experiments. PTBC, TTTN and TTTT conducted experiments. PTBC, HDN analyzed data. PTBC, HDN and WS wrote the manuscript. All authors read and approved the manuscript.
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Chu, P.T.B., Phan, T.T.P., Nguyen, T.T.T. et al. Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis. World J Microbiol Biotechnol 39, 143 (2023). https://doi.org/10.1007/s11274-023-03566-8
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DOI: https://doi.org/10.1007/s11274-023-03566-8