Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins

Lam, K. H. E.; Kc, Chow; Wong, Wan Keung

doi:10.1016/s0168-1656(98)00041-8

Cited by 81 publications

(41 citation statements)

References 31 publications

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“…Strains of species like B. licheniformis and B. amyloliquefaciens have been used for the production of various proteins of homologous origin, such as proteases, lipases, and starchdegrading enzymes. Some species, especially B. subtilis, have also been used as hosts for the production of heterologous proteins (1,16,18). Genetic tools for efficient expression and secretion of recombinant proteins in the latter organism have been rapidly developed and successfully used (10,23).…”

Section: Discussionmentioning

confidence: 99%

Surface Display of Recombinant Proteins onBacillus subtilisSpores

Isticato¹,

Cangiano²,

Trần³

et al. 2001

J Bacteriol

202

235

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We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescentactivated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 ؋ 10 3 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

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Section: Discussionmentioning

confidence: 99%

Surface Display of Recombinant Proteins onBacillus subtilisSpores

Isticato¹,

Cangiano²,

Trần³

et al. 2001

J Bacteriol

202

235

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“…B. subtilis is highly appreciated for biotechnological applications because it has a large capacity to secrete high-quality proteins into the culture medium and because it has the status of generally recognized as safe (18,38,50). Furthermore, B. subtilis is amenable to genetic engineering, and many expression systems are available (2,16,31,40,43,44). This prompted us to investigate whether the secretion machinery of B. subtilis, which is also involved in membrane protein biogenesis (52), can be exploited for membrane protein overproduction.…”

mentioning

confidence: 99%

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

Zweers

Wiegert

Dijl

2009

Appl Environ Microbiol

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Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the W regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

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“…Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes. B. licheniformis bears the GRAS (generally regarded as safe) status (Lam et al 1998). The availability of the genome sequence from Bacillus licheniformis ATCC 14580 facilitates direct genetic manipulations and thus maximum exploitation (Waldeck et al 2007).…”

mentioning

confidence: 99%

Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3)

et al. 2011

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A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50• C and pH 6. Km and Vmax were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy Ea, enthalpy of activation ∆H and entropy of activation ∆S were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pKa1 and pKa2 of the ionizable groups of active site residues involved in Vmax were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.

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Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins

Cited by 81 publications

References 31 publications

Surface Display of Recombinant Proteins onBacillus subtilisSpores

Surface Display of Recombinant Proteins onBacillus subtilisSpores

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3)

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