Abstract
Bacillus subtilis is a generally regarded as safe (GRAS) microorganism, which has been used for industrial production of recombinant enzymes. Many inducible and inducer-free expression vectors have been developed for intracellular production; some of those demonstrated the capability for protein expression up to 42% of total cellular proteins. In this chapter, we introduce the method to enhance the expression of soluble protein in B. subtilis. It includes the construction of vectors, the transformation of a plasmid into B. subtilis, and checking the expression of the protein.
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References
Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62:137–189. https://doi.org/10.1016/S0065-2164(07)62006-1
Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17. https://doi.org/10.1139/w03-076
Nguyen HD, Nguyen QA, Ferreira RC, Ferreira LCS, Tran LT, Schumann W (2005) Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54:241–248. https://doi.org/10.1016/j.plasmid.2005.05.001
Phan TTP, Nguyen HD, Schumann W (2006) Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46:189–195. https://doi.org/10.1016/j.pep.2005.07.005
Nguyen HD, Phan TTP, Schumann W (2007) Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis. Curr Microbiol 55:89–93. https://doi.org/10.1007/s00284-006-0419-5
Phan TTP, Nguyen HD, Schumann W (2013) Construction of a 5′-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis. J Biotechnol 168:32–39. https://doi.org/10.1016/j.jbiotec.2013.07.031
Phan TTP, Tran LT, Schumann W, Nguyen HD (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Factories 14:72. https://doi.org/10.1186/s12934-015-0255-z
Phan TTP, Nguyen HD, Schumann W (2012) Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. J Biotechnol 157:167–172. https://doi.org/10.1016/j.jbiotec.2011.10.006
Tran DTM, Phan TTP, Huynh TK, Dang NTK, Huynh PTK, Nguyen TM, Truong TTT, Tran TL, Schumann W, Nguyen HD (2017) Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis. Microb Cell Factories 16:130. https://doi.org/10.1186/s12934-017-0747-0
Schulz A, Schumann W (1996) hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J Bacteriol 178:1088–1093. https://doi.org/10.1128/jb.178.4.1088-1093.1996
Saito H, Shibata T, Ando T (1979) Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg. Mol Gen Genet MGG 170:117–122. https://doi.org/10.1007/BF00337785
Popham DL, Setlow P (1993) Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase. J Bacteriol 175:2917. https://doi.org/10.1128/jb.175.10.2917-2925.1993
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28. https://doi.org/10.1016/0378-1119(90)90336-p
Phan T, Huynh P, Truong T, Nguyen H (2017) A generic protocol for intracellular expression of recombinant proteins in Bacillus subtilis. Methods Mol Biol 1586:325–334. https://doi.org/10.1007/978-1-4939-6887-9_21
Acknowledgments
This research is funded by Vietnam National University, Ho Chi Minh City, Vietnam (VNU-HCM) under grant number B2019-18-10.
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Nguyen, H.D., Phan, T.T.P. (2022). A Protocol to Enhance Soluble Protein Expression in the Cytoplasm of Bacillus subtilis. In: Garcia Fruitós, E., ArÃs Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_14
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DOI: https://doi.org/10.1007/978-1-0716-1859-2_14
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