Bacillus subtilis as a production host for membrane protein SpoIISA
Ľudmila Vavrová, Stanislava Rešetárová, Patrik Florek, Katarína Muchová and Imrich Barák
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava 45, Slovakia, E-mail: ludmila.vavrova@savba.sk
Gram-positive bacterium Bacillus subtilis has great potential as the host for production of cytoplasmic proteins, protein complexes and membrane proteins assigned for structural and functional analysis.
As a model for studying membrane proteins we have chosen B. subtilis protein complex SpoIISA – SpoIISB. These proteins belong to bacterial programmed-cell death system, forming a toxin-antitoxin couple. SpoIISA as a toxin has three putative membrane-spanning helices. The remaining two-thirds of the protein are predicted to be localized in the cytoplasm. SpoIISB antitoxin is a small cytosolic protein and it was found, that SpoIISB makes complex with cytosolic part of SpoIISA.
In order to choose the most productive
expression system for production of SpoIISA as a membrane protein we have
developed its expression in B. subtilis. We were
testing different possible expression systems, namely xylose inducible,
IPTG inducible and subtilin-regulated system, respectively. We have compared
the strength and leakiness of all three systems. Western blot analysis revealed
that the highest level of expression was obtained in SURE system (~ 10 - 100 μg protein/100 ml).
To further study localization of SpoIIS system in B. subtilis cells we have prepared fusion of transmembrane domain of SpoIISA, under control of its own promoter, with GFP.
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Acknowledgements.
This
work was supported by the grant APVT-51-027804, No. ESF-EC-0106, LPP-0218-06 and
VEGA grant 2/7007/27 from
the Slovak Academy of Sciences.