Isolation of the recombinant PsbO protein from higher plants

Daryna Kulik1, 2, Ivana Kuta Smatanova1, 3 and Jaroslava Kohoutova1

 

1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and School of complex systems, Zamek 136, 373 33 Nove Hrady

2 University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 37005 Ceske Budejovice

3 Academy of Sciences of the Czech Republic, Inst. of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady

 

Photosystem II (PSII) is a large protein complex in a thylakoid membrane of higher plants, algae and cyanobacteria that catalyses light-driven oxidation of water and produces molecular O2, electrons and protons. The water splitting and oxygen release occur in the catalytic centre of PSII – the oxygen-evolving centre (OEC) that contains manganese-calcium cluster (4:1 Mn:Ca) located close to the luminal surface of the transmembrane domain and surrounded by intrinsic and extrinsic components in thylakoid membranes. PsbO (33kDa), PsbP (23kDa), PsbQ (17 kDa), PsbR (10 kDA) are extrinsic proteins associated to the luminal side of PSII in higher plants, which maintain stability of water oxidation site and correct ionic environment during water oxidation [1, 2, 3]. The knowledge of the structure of PSII and its proteins plays an important role in understanding of the water splitting.

The objective of the project is to obtain recombinant PsbO protein from Pisum sativum and Spinacia oleracea. The extraction of mRNA from leaves and synthesis of cDNA are the first steps to get the psbO gene. The conditions for polymerase chain reaction (PCR) were optimized and DNA fragments encoding the psbO gene were obtained. pET-28b(+) vector was used for overexpression of recombinant PsbO/HisPsbO proteins. Purification, crystallization and structure determination of the recombinant proteins PsbO/HisPsbO of the oxygen-evolving complex from Spinacia oleracea and Pisum sativum are underway.

 

This research was supported by the ME CR (COST LD11011, CZ.1.05/2.1.00/01.0024), by the AS CR (AV0Z60870520) and GAJU 170/2010/P for DK.

 

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2.       Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Architecture of the photosynthetic oxygen-evolving center. Science 303:1831–1838. doi:10.1126/science.1093087

 

3.       Loll B, Kern J, Saenger W, Zouni A, Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 A ° resolution structure of photosystem II. Nature 438:1040–1044. doi:10.1038/ nature04224