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Engineering of the cellular quality control systems in bacillus subtilis for the production of high value-added proteins

Deliverables

Gram-positive bacteria of the species Bacillus are well known for the high levels of secretion of several of their homologous proteins. Attempts to secrete heterologous proteins at high levels have, however, often met with little success. An important result from this project was that mutations in the dlt operon, responsible for the alanylation of cell wall teichoic acids, resulted in a significant increase in the production of a number of secretory proteins, including Bacillus amylases, and the protective antigen (PA) of Bacillus anthracis. This finding holds promise for the improved secretion of other (heterologous) proteins. The results have been published in the international scientific literature and a patent was filed.
A total of six integrational plasmid vectors were constructed for use in Bacillus subtilis. These vectors allow addition of two different kinds of tags to the C-terminal end of any protein encoded by the B. subtilis chromosome. In one case, an epitope tag is added (cMyc, HA, FLAG)which allows detection of proteins by commercially available antibodies. This method can be used for affinity purification of proteins of interest and for their detection in Western blots. In the other case, a localisation tag is added to the proteins of interest (GFP, CFP, YFP) allowing their detection within one of the compartments of the B. subtilis cell. In addition, these tagged proteins can be detected in Western blots. The results have been published (Kaltwasser et al., 2002, Appl. Environm. Microbiol. 68, 2624-2628).