ScholarWorks@UNO

Synthesis of Functional Recombinant Subunits of Phycoerythrin II

Cyanobacteria possess light-harvesting complexes called phycobilisomes (PBS). Chromophores, called bilins, direct light to the chlorophyll-containing reaction centers for photosynthesis. These bilins are attached to the phycobiliproteins by lyases at specific cysteine sites. The focus of this project, Synechococcus RS 9916, is a marine species of cyanobacteria that lives deep in the water column and can undergo chromatic acclimation, where it can alter its pigments to best absorb the blue or the green light available. It uses orange-colored phycourobilin and red-colored phycoerythrobilin to capture blue and green light, respectively. The proteins of interest, MpeA and MpeB, are the alpha and beta subunits of phycoerythrin II, which is the outermost protein in the rod in the PBS complex. The goal was to produce and test a construct that contains both mpeA and mpeB in one plasmid; each protein will be fused with a hexa-histidine tag (Ht), to allow for easy purification. The clones were transformed into E. coli to overexpress the proteins of interest in E. coli, which was verified by SDS-PAGE. Preliminary results suggest protein production is occurring. The most productive clones were then co-expressed in E. coli with known and putative lyases and bilin synthesis genes to validate the clones. Though mpeA and mpeB have been cloned and expressed separately, the goal is to produce these alpha and beta subunits together, as they exist in cyanobacteria—for use in further research.