Photosynthetica 2009, 47(3):355 | DOI: 10.1007/s11099-009-0056-x

Expression, purification and preliminary crystallization study of RpaC protein from Synechocystis sp. PCC6803

E. Cséfalvay1,*, M. Lapkouski1, O. Komárek2
1 Department of Structure and Function of Proteins, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic and Institute of Physical Biology, University of South Bohemia in České Budějovice, Nové Hrady, Czech Republic
2 Department of Biological Dynamics, Laboratory of Ecology and Physiology of Plants, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic and Institute of Physical Biology, University of South Bohemia in České Budějovice, Nové Hrady, Czech Republic

State transitions in cyanobacteria are physiological adaptation mechanisms that change the interaction of the phycobilisomes with the photosystem I and photosystem II core complexes. This mechanism is essential for cyanobacteria at low light intensities. Previous studies of cyanobacteria have identified a gene named rpaC, which appears to be specifically required for state transitions. The gene product of rpaC is very probably a transmembrane protein that is a structural component of the phycobilisome-photosystem II supercomplex. However, the physiological role of RpaC protein is unclear.
Here we report the construction of an expression system that enables high production of fusion protein TrxHisTagSTag-RpaC, and describe suitable conditions for purification of this insoluble protein at a yield of 3 mg per 1 dm3 of bacterial culture. Cleavage with HRV 3C protease to remove the TrxHisTagSTag portion resulted in low yields of RpaC-protein (∼ 30 µg/dm3 of bacterial culture), therefore the applicability to structural studies was tested for the fusion protein only. Several preliminary conditions for crystallization of TrxHisTagSTag-RpaC were set up under which microcrystals were obtained. This set of conditions will be a good starting point for optimization in future crystallization experiments. TrxHisTagSTag-RpaC protein may prove useful in biochemical studies where the small size of RpaC protein is limiting the investigation of interactions with significantly larger parts of the photosynthetic apparatus. Furthermore, the purification procedure described here might also be applied to the production and purification of other small membrane proteins for biochemical and structural studies.

Additional key words: affinity chromatography; photosystem; phycobilisome - PSII supercomplex

Received: August 12, 2008; Accepted: May 25, 2009; Published: September 1, 2009  Show citation

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Cséfalvay, E., Lapkouski, M., & Komárek, O. (2009). Expression, purification and preliminary crystallization study of RpaC protein from Synechocystis sp. PCC6803. Photosynthetica, 47(3), 355. doi: 10.1007/s11099-009-0056-x
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