Abstract:
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.