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Overproduction of membrane protein overproduction in Bacillus subtilis

21 March 2012

PhD ceremony: Ms. J.C. Zweers, 12.45 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Overproduction of membrane protein overproduction in Bacillus subtilis

Promotor(s): prof. J.M. van Dijl

Faculty: Medical Sciences

Bacterial membrane proteins perform many essential tasks within the cell and are potential targets for antimicrobial therapies. Compared to other proteins, research on membrane proteins has been lagging behind. This is mainly due to the hydrophobic nature of these proteins, which interferes with their overproduction, purification and crystallization. This thesis describes the development of the Gram-positive microbial cell factory Bacillus subtilis as a host for membrane protein overproduction and subsequent purification. Two stress-responsive mechanisms within B. subtilis were identified that pose direct bottlenecks for membrane protein overproduction: the σW regulon and the CssRS two-component system. Accordingly, deletion of these stress-responsive systems drastically improves membrane protein overproduction yields. Therefore, the genes in these regulons and their interactions were accurately charted, with special focus on membrane-bound proteases involved in these two stress-responsive systems. The results show that the induction of membrane protein overexpression elicits a carefully balanced stress-responsive network that either permits or counteracts the actual membrane protein production. The balance can be shifted upon interference with specific stress responses. This is illustrated by the observation that deletion of relevant stress-responsive systems can interfere with membrane protein overproduction due to the activation of other stress-responsive systems, whereas mild inhibition of the same stress-responsive systems improves the capacities to overproduce membrane proteins. Based on these results it is now possible to use B. subtilis for the production and functional characterization of membrane proteins from important pathogenic bacteria, like Staphylococcus aureus, which is dearly needed for the development of novel antimicrobial therapies.

Last modified:13 March 2020 01.01 a.m.
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