Purification and crystal structural analysis of the blue fluorescent protein of Vibrio vulnificus
| Autor: | Ming-Chun Hung, 洪銘駿 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 The blue fluorescent protein (BFP), which was isolated from Vibrio vulnificus CKM-1, exhibits homology to the family of short-chain dehydrogenase/reductase (SDR). This protein has fluorescence spectra with two excitation peaks at 283 and 352 nm, and one emission peak at 456 nm. Our previous studies revealed that the fluorescence intensity of G176S-BFP mutant is about two-folds higher than wild-type BFP (WT-BFP). In this study, we tried to use bio-crystallography to build the molecular structures of WT-BFP and G176S-BFP and to elucidate the fluorescence mechanism of BFP. The coding sequences of WT-BFP and G176S-BFP mutant were cloned into vector pET21b, respectively. Both the WT-BFP and G176S-BFP recombinant proteins were expressed in E. coli BL21 (DE3), which were subsequently purified by nickel affinity column and gel-filtration column. For co-crystallization, a concentration of 7 mg/ml of WT-BFP and G176S-BFP were mixed with 2 mM NADPH, respectively, and then, crystals of WT-BFP/NADPH (Cryo II 18) and G176S BFP/NADPH (PEG/Ion screen 23) were obtained by the sitting-drop vapor diffusion method. The resolutions are 3 Å and 1.2 Å, for WT-BFP/NADPH and G176S BFP/NADPH, respectively. Hence, only G176S-BFP 3D structure has been built because the resolution of WT-BFP is low. The structure of G176S-BFP is similar to the modeled structure compared with previous study. Furthermore, the mechanism of BFP fluorescence is going to clarify by molecular structures. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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