Cloning and characterization of a blue fluorescent protein from Vibrio vulnificus

The blue fluorescent protein (BFPVV) gene bfpvv from Vibrio vulnificus CKM-1 was cloned and sequenced. The transformants exhibited blue fluorescence when irradiated by UV source. Nucleotide sequence analysis predicted an ORF of 717 bp encoding a 239-amino-acid polypeptide with a calculated molecular mass of 25.8 kDa. The nucleotide sequence of the bfpvv gene and its deduced amino acid sequence showed significant homology to those of the short-chain dehydrogenase/reductase (SDR) family proteins from various organisms. Some functionally important residues in SDR were strictly conserved in BFPVV, such as an active-site Tyr145, a catalytic site Lys149, and a common GlyXXXGlyXGly pattern in the N-terminal part of the molecule. By changing three amino acid residues, Tyr145, Lys149, and Gly9 to Phe, Ile, and Val, respectively, it was found that the G9V mutant did not generate blue fluorescence, while mutants Y145F and K149I have 126 and 68.5% fluorescence compared with the wild-type BFPVV.