Expression of red fluorescent protein All1280 GAF2 in E. coli and constructing mutation of cyanobacteriochrome

Cyanobacteriochromes contain GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains in the N-terminal region that bind phycocyanobilin autocatalytically. In the current study, we amplified the cyanobacteriochrome gene fragment of all1280 gaf2 from Anabaena sp. PCC 7120 using PCR, and then inserted it into pET-30a(+). For over-expression, both pET-all1280 gaf2 and pACYC-ho1-pcyA, which catalyze phycocyanobilin (PCB) biosynthesis, were transformed into E. coli BL21 (DE3). Cells harboring pET-all1280 gaf2 and pACYC-ho1-pcyA expressed chromophorylated All1280 GAF2 successfully. Results showed that All1280 GAF2 underwent reversible photoconversion between the 15E form (λ_max=560 nm) and 15Z form (λ_max=413 nm). Using fluorescent microscopy, we detected a red fluorescence/no fluorescence reversible photoconversion of All1280 GAF2 in E.coli BL21(DE3). Cys53 was essential for photoconversion of All1280 GAF2 because its mutagenesis resulted in a PCB adduct, which exhibited no photoconversion but stable red fluorescence. Compared with the wild-type, All1280 GAF2(C53A) had stronger red fluorescence with higher extinction coefficients and fluorescence yields. It is expected that these two constructs could serve well in the labeling of living cells.