Improvement of the PSII purification procedure for crystallization
Olga Shmidt1,2, Jaroslava Kohoutova1, Estela Pineda
Molina4, Jose A. Gavira4, Pavlina
Rezacova5, David Kaftan2, Michal Kuty1,3, Juan Manuel Garcia-Ruiz4 and Ivana
Kuta Smatanova1,3
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
4Laboratorio de Estudios
Cristalográficos, Edf. López Neyra, P.T. Ciencias de la Salud, Avenida del
Conocimiento s/n, 18100 Armilla (Granada) Spain
5 Institute of Organic Chemistry & Institute of Molecular Genetics of the Academy of Science of the Czech Republic, v.v.i., Flemingovo n. 2, 16637 Prague, Czech Republic
The aim of this work is to develop purification procedure for higher plant PS II providing material for its crystallization and high resolution X-ray diffraction.
We have re-designed the standard protocol and implemented novel procedures for isolation of the PS II enriched thylakoid membranes from the plant material. Handpicked leaves of the Pisum sativum L. were homogenised and at the first step highly active thylakoid membranes were isolated. Then, sucrose density gradient ultracentrifugation and ion exchange chromatography techniques were used for purification of the PSII complexes from the solubilised thylakoid membranes. The complexes’ activity was followed throughout the isolation routine with absorption and fluorescence spectroscopy. The purity of the samples was assessed by optical spectroscopy and western blotting.
Purification procedure that included the ion exchange chromatography preceding the ultracentrifugation yielded highly pure sample. Unfortunately the quantity of the sample was not enough for crystallization trials.
We currently continue to optimize the protocol for PSII complex purification. Methods and approaches for crystallisation of both membrane and soluble proteins will be implemented subsequently.
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 VS.