| Summary: | 碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 100 === Fluorescent proteins (FPs), like green fluorescent protein(GFP) and its derivatives, have been used for various applications as in detecting protein expression and conducting functional studies in living systems. Moreover, complex functional studies can be performed using FPs such as visualizing protein-protein interactions in living cells by FRET (Fluorescence Resonance Energy Transfer) detection technique. Although GFP and its variants have become an invaluable tool in the field of non-invasive in vivo imaging technology, a major drawback of all members of GFP family-their strict requirement for molecular oxygen as a cofactor for the synthesis of their respective chromophores. In contrast to GFP, our laboratory developed blue fluorescent protein-dBP6, which emits blue fluorescence under normoxia and hypoxia due to its cofactor NADPH as the fluorescent chromophore. To further extend the use of dBP6, we utilized a FMN (flavin mononucleotide) based green fluorescent protein iLOV (≈11kDa) that may potentially become FRET pair with dBP6 under hypoxia since its illuminated mechanism is similar to that of dBP6. In addition, studies regarding to iLOV application in mammalian cells have not been explore yet. Our goal is to evaluate whether iLOV can be utilized for protein labeling and detection under hypoxia and normoxia in mammalian cells. By changing the iLOV codon usage, we successfully expressed iLOV in mammalian cells under normoxia and hypoxia and found that it can function as a fusion protein. After checking the spectrum of iLOV and dBP6, we found the overlaping region between iLOV maximum excitation peak and dBP6 emission peak. Next, we linked the dBP6 and iLOV together to further investigate FRET application in cells. However, the results showed that iLOV could be excited at the spectra of 350nm overlapped with that of dBP6(350nm). In order to solve this problem, we measured different excitation wavelengths of dBP6 and iLOV every 10nm in between 320nm to 350nm. The result indicated that lowering the excitation wavelength of dBP6 might avoid the above problem of iLOV. Due to the results of excitation wavelength shift, we can design the filter specific for the wavelength below 330nm for further application under hypoxia.The fluorescent intensity still have to be optimized. This study provides us a new direction of detecting protein protein interaction under hypoxia.
|
|---|
