MOLECULAR STUDIES OF FLUORESCENT PROTEIN GENE bfgV FROM Vibrio Vulnificus CKM-1

• Main Authors: Chun-Chin Chang, 張淳欽
• Other Authors: Ming-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/62548682928695074919

博士 === 國立成功大學 === 基礎醫學研究所 === 93 === Abstract 　Blue fluorescent protein BfgV, belonging to the SDR family, was found in non-bioluminescent pathogen Vibrio vulnificus CKM-1. This protein had two excitation peaks at 283 nm and 352 nm respectively, and one emission peak at 456 nm. The results of HPLC analysis and L-glutamic dehydrogenase reaction indicated that BfgV fluoresced through augmenting 10 times the intrinsic fluorescence of NADPH binding on it. Comparison of gene organizations around bfgV and two analogues in Vibrio cholera El Tor N16961 and Shewanella oneidensis MR-1 revealed that each of them had a transcriptional regulatory gene located at the vicinal upstream region. Insertional disruption of the upstream regulatory gene bfgR and electrophoretic mobility shift assay proved that bfgV was negatively regulated by bfgR. In the directed evolution process, wild type bfgV gene was subjected to three cycles of error-prone PCR and one cycle of DNA shuffling. A prominent mutant D7 displayed 4 times the fluorescent intensity of BfgV. The emission peak of D7 was at 440 nm, 16 nm shorter than that of BfgV, but the excitation peaks of these two proteins were the same. D7 possessed eight amino acid substitutions while only seven contributed positive influence on it. After assigning these mutations to the modeled 3D-structure of BfgV, we found three of them appeared at loop 4 and loop 6, which comprised the NADPH binding site. This unusual mutant cluster suggested these regions played a key role on fluorescent intensity of BfgV. In addition, D7 synthesis and fluorescent expression in E. coli transformants were nearly synchronic. This property was quite different from that of GFP.