Cloning, expression of the psbU gene, and functional studies of the recombinant 12-kDa protein of photosystem II from a red alga Cyanidium caldarium

The encoding extrinsic 12-kDa protein of oxygen; evolving PS II complex fro m a red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure. The gene encodes a putative polypeptide of 154 amino acids with a calculated molecular mass of 16,714 Da. The full sequence of the protein includes two characteristic transit peptides, one for transfer across the chloroplast envelope and anot her for targeting into the thylakoid lumen. This indicates that the protein is encoded in the nuclear genome. The mature protein consists of 93 amino acids with a calculated molecular mass of 10,513 Da. The cloned gene was su ccessfully expressed in Escherichia coli and the resulting protein was puri fied, reconstituted to CaCl2-washed PS II complex together with the other e xtrinsic proteins of 33 and 20 kDa and cyt c-550. The recombinant 12-kDa pr otein bound completely with the PSII complex, which resulted in a restorati on of oxygen evolution equal to the level achieved by binding of the native 12-kDa protein. (C) 1999 Academic Press.