Abstract:
To construct pET-
alr1966gaf2, a fragment of
alr1966gaf2 was amplified by polymerase chain reaction (PCR) from
Anabaena sp. PCC7120, and then inserted into pET-30a(+). For over-expression, pET-
alr1966gaf2 was transformed into
Escherichia coli BL21(DE3) containing pACYC-
ho1-
pcyA and biliproteins were co-expressed successfully. Results showed that bili-Alr1966GAF2 had a sequential reversible photoconversion in three different states. We also detected red fluorescence reversible photoconversion of Alr1966GAF2 in 15E-P
514 nm/15Z-P
428 nm forms. Via site-directed mutagenesis, we mutated conserved Cys into Ala in the conserved DXCF motif of Alr1966GAF2, resulting in Alr1966GAF2(C72A). Alr1966GAF2(C72A)-PCB showed strong red fluorescence and high fluorescence quantum yield of 0.11. Furthermore, Alr1966GAF2(C72A) could bind to phycoerythrobilin (PCB) and biliverdin (BV) covalently, with strong red fluorescence. Therefore, as a red fluorescent protein, Alr1966GAF2(C72A) could be further developed as a fluorescent probe and applied in life sciences.