PARENT SESSION
Posters P5A Type II reaction centres : Structure. Abstracts (289-312)

Cloning, overexpression and purification of polypeptide subunits of the cytochrome b559 from higher plants. Rafael PICOREL^*,1, Maria Angeles LUJAN^*,1, Miguel ALFONSO^*,1, Inmaculada YRUELA^*,1, ¹ Aula Dei Experimental Station-CSIC, Zaragoza, Spain

ABSTRACT- The cytochrome (Cyt) b559 is a membrane-bound protein wich is a constituent of the photosystem II reaction center but with unknown function. The cytochrome is made of two polypeptide subunits, alpha and beta. The alpha-subunit (9 kDa) is encoded by the psbE gene and the beta-subunit (4.5 kDa) by the psbF gene. Both genes are of chloroplastic origin and each polypeptide spans the thylakoid membrane with a single alpha-helix. The two polypeptides are bound by a heme group coordinated by two histidines, one from each polypeptide. Our work is aiming to obtain in vitro reconstitution of the Cyt b559 for future structural and functional studies. To that end we have cloned and overexpressed separately both Cyt b559 subunits from sugar beet in E. coli as a fusion protein with the maltose-binding protein (MBP) using the pMALc2x vector modified with His6-tag. Since membrane proteins normally pose formidable challange to overexpress in E. coli we have taken special attention to obtain significant amounts of the fusion proteins in a soluble form. After bacterial sonication, the resultant supernatant was first purified by Ni²⁺-IMAC and then treated with trombine protease to release the Cyt b559 subunits from the fusion protein. The Cyt b559 subunits were then further purified by ion-exchange chromatography and subsequently by size-exclusion chromatography. The protein subunits were identified by SDS-PAGE and in the case of the alpha-subunit also by immunodetection.

KEY WORDS: cytochrome b559, photosystem II, chromatography, immunodetection