Cloning and expression of a cDNA encoding the extrinsic 20 kDa protein of photosystem II from a red alga Cyanidium caldarium

Abstract

Oxygen-evolving PS II complex purified from a red alga, Cyanidium caldarium, contains four extrinsic proteins; they are 12 kDa, 20 kDa, 33 kDa proteins and cytochrome (cyt) c-550 [Enami et al. (1995) B.B.A. 1232, 208 - 216]. In this study, the 20 kDa protein was cloned and expressed in E. coli. The 20 kDa protein was purified from CaCl2-treated extracts of purified PS II complex and its N-terminal amino acid sequence was determined. Oligonucleotides synthesized on the basis of its N-amino acid sequence were used as primers for degenerated PCR, and complete sequence of the gene was determined using modified 5'- and 3'- RACE methods. The resulted open reading frame encodes a protein of 218 amino acid residues of which, the first 62 residues were assigned as leader sequences according to the N-terminal sequence of the mature protein. The resulted mature protein contains 146 amino acid residues with a molecular mass of 16386 Da. A search with the Swiss-Prot database revealed that the Cyanidium caldarium 20 kDa protein is 36.3% and 30.3% identical to the extrinsic 17 kDa protein from Volvox and Chlamydomonas, respectively, suggesting that the red algal 20 kDa protein is possibly an ancestor of the 17 kDa protein in green algae. An expression vector containing the 20 kDa gene was constructed in pCAL-n-EK plasmid, and the successful expression of the protein has been confirmed. We are currently performing reconstitution experiments with the recombinant 20 kDa protein to CaCl2-washed red algal PS II to study the binding and functional properties of the protein.