Synechocystis sp. PCC 6803 plastocyanin: Cloning and regulatory studies, and forced expression of Silene pratensis plastocyanin in Escherichia coli.

Abstract

The backbone of biological energy transduction is the transfer of electrons through proteins that in many cases contain a metal cofactor. Platocyanin (a soluble, low molecular weight copper protein), functions as a redox carrier in the photosynthetic electron transport chain. The overall goal of this research project was to develop site specific mutants of the blue copper protein plastocyanin to delineate the roles particular residues play in facilitating electron transfer. Toward this goal, the isolation and sequencing of the first unicellular cyanobacterial (Synechocystis sp. PCC 6803) plastocyanin gene (petE) is presented. Plastocyanin was detected in Synechocystis sp. PCC 6803 when 3 $\mu$M copper was added to the growth medium, BG-11. The petE gene was cloned from a genomic $\lambda$ EMBL 3 library by screening with the petE gene from Anabaena sp. PCC 7937. The Synechocystis 6803 petE gene is present as a single copy and, as deduced from the DNA sequence, encodes a precursor protein of 126 amino acids. The 29 amino acid transit peptide, thought to direct the plastocyanin-precursor to the thylakoid lumen, shows substantial homology to the Anabaena 7937 transit peptide. The mature protein has the greatest homology (61%) to the green alga Scenedesmus obliquus plastocyanin. Despite the low overall homology, the copper binding ligands and certain aromatic residues remain highly conserved. Northern hybridization analysis indicates that the steady state petE mRNA level was independent of the copper concentration in the growth medium; therefore this gene is not transcriptionally regulated. A heterologous expression system was developed for the Silene pratensis petE gene in Escherichia coli by inserting the coding region into a bacterial expression vector (pUC19) containing the lac promoter and a bacterial ribosome binding site. A fusion protein ($\sim$18,500 daltons) was detected by immunoblotting techniques after induction of transcription using isopropyl thiogalactoside (IPTG). The recombinant polypeptide product is not processed or secreted by E. coli and is sequestered into inclusion bodies.