Fluorescent intensity of a novel NADPH-binding protein of Vibrio vulnificus can be improved by directed evolution.
Blue fluorescent protein, BfgV, found from Vibrio vulnificus CKM-1, fluoresces through augmenting the intrinsic fluorescence of bound NADPH. Random mutagenesis and DNA shuffling were applied to increase the fluorescent intensity of BfgV. The wild type bfgV gene was subjected to four cycles of mutagenesis processes. A prominent D7 mutant protein had fluorescent intensity four times larger than wild type BfgV. The emission wavelength of this mutant protein appeared at 440 nm, which was 16 nm shorter than that of BfgV. There were eight amino acid substitutions in D7. As these substitutions were assigned to the modeled 3D structure of BfgV, three of them, V83M, G176S, and E179K, were shown to be located around NADPH-binding site. Time course analysis indicated the synthesis of D7 protein and fluorescent expression in Escherichia coli transformants were synchronic. This property was different from that of wild type GFP.
Chang
Chun Chin
CC
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.
Chuang
Yin Ching
YC
Chang
Ming Chung
MC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003198:Computer Simulation; D032621:DNA Shuffling / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009249:NADP / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D041261:Vibrio vulnificus / Q000235:genetics* / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D009249:NADP