Cloning and Expression of the cpeS gene from cyanobacteria into an expression plasmid to allow for the production of the fluorescent protein phycoerythrin inside E. coli.

Cloning and Expression of the cpeS gene from cyanobacteria into an expression plasmid to allow for the production of the fluorescent protein phycoerythrin inside E. coli. / / Melody Bolton, Christina M. Kronfel and Wendy M. Schluchter / / Cyanobacteria produce phycoerythrin (PE), a red fluorescent protein used to absorb green light to perform photosynthesis. The long term goal of this research is to use its fluorescent properties as a reporter of protein localization and gene expression in living cells. PE \u27s fluorescent properties and characteristic red color are due to the attachment of phycoerythrobilin (PEB) at five different sites on cysteine (Cys) residues. Cysteines contain a sulfur side chain allowing for PEB to bind covalently forming a thioether bond. The attachment of PEB to PE is catalyzed by enzymes called bilin lyases. This study focuses on the bilin lyase CpeS from the cyanobacterium Fremyella diplosiphon (FD CpeS) that attaches PEB to cysteine at sequence position 80 on the beta subunit of phycoerythrin (CpeB; Biswas, et al. 2011). CpeB has three chromophore attachment sites at positions cysteine-80, Cysteine-165, and one PEB attached at two cysteines located at 48 and 59. The biosynthetic pathway for this subunit of PE can be recreated in the bacterium E. coli by cloning all of the genes hypothesized to be involved in this process and testing whether this red fluorescent protein is produced. The purpose of this project was to clone the gene encoding CpeS into an expression plasmid (pCDFDuet) to be used in experiments to test the hypothesis that other genes encode lyases for the other bilin attachment sites. / / Biswas, A., et al., Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481. Journal of Biological Chemistry, 2011. 286(41): p. 35509-35521. / / Funding: UNO COSURP and NS