Reconstitution in vivo and Photochemical Properties of Several Cyanobacteriochrome Domains from Thermosynechococcus elongatus BP-1

By protein sequence homology comparison with the Pb/Pg-type cyanobacteriochrome TePixJ and TeTlr0924, three homologous genes tlr0911, tlr1215 and tlr1999 from Thermosynechococcus elongatus BP-1 were found. By molecular cloning techniques, the GAF domains of those genes were cloned into expression vector pET30a(+), respectively. The E.coli strain BL21 (DE3) harboring the expression plasmid and the pACYCDuet-ho1-pcyA plasmid for phycocyanobilin (PCB) were induced to generate recombinant proteins. The expressed proteins (His)₆ tagged at the N-terminus were purified with nickel-affinity His-Trap chelating column. The purified proteins were identified with zinc-induced fluorescence, acidic urea denaturation, fluorescence and absorption spectrum. Results showed that Tlr0911-GAF contained two covalently bound bilin chromophores, phycoviolobilin (PVB) and phycocyanobilin (PCB), and exhibited reversible photoconversion between a blue-absorbing form at 406 nm (Pb_{406 nm}) and a green-absorbing form at 527 nm (Pg_{527 nm}). Tlr1999-GAF was also covalently bound with PVB and PCB, and reversible photoconversion existed between a blue-absorbing form at 417 nm (Pb_{417 nm}) and a teal-absorbing form at 496 nm (Pt_{496 nm}). Neither Tlr1215-GAF1 nor Tlr1215-GAF2 could be spontaneously bound with bilin chromophore.