| The zinc ion is an important metal ion in cellular activities and a special signal ion in cells. It participates in a number of physiological and biochemical functions in cells and plays an important role in human health. The fluorescent probes have a broad prospect in the study of physiological effects caused by the ion. The fluorescent probes for zinc ions have been designed with small chemical molecules nowadays. Although they have been used for the detection an imaging of zinc ions in living cells, they have lots of defects, such as difficulties of making them into living cells, no special target functions, influences on the normal cells. And the fluorescent protein probe can solve these problems. It can be easily transfected into living cells, target to specific sites with genetic engineering methods in vivo, and less influence on the physiological function of the living cells.The fluorescent protein probe based on fluorescent resonance energy transfer (FRET) technology can be used to detect and image real-time signal transduction processes of the living cells. Cameleon is a typical probe for calcium, but zinc ion probe is still in the initial stage of development. In order to facilitate the research dynamic signal processes of zinc ion in living cells, we constructed fluorescent protein-based probe for zinc ion detection based on FRET technology. We also probe the zinc ion dynamics both in the environment and living cells using our designed probes.Firstly, a subunit of MT2A protein was connected with a pair of fluorescent proteins ECFP and cpCitrinel74 (circular permutated YFP) which can produce FRET. GGS and GGSGG were used for connecting peptides. The plasmids pET28a (+) and pCDNA3.1 (+) were used as the expression vectors. The plasmid pET28a (+)-ECFP-GGS-MT2Aa-GGSGG-cpCitrine174 was transformed into E.coli BL21 (DE3), and then induced by IPTG. The recombinant protein His-ECFP-GGS-MT2A-GGSGG-cpCitrinel74 was purified through Ni+ affinity chromatography and experimented in ITC and fluorescence spectrometer after adding Zn2+in vitro. The plasmid pCDNA3.1(+)-ECFP-GGS-MT2Aa-GGSGG-cpCitrine174 was transfected into the HeLa cells, and the fluorescence images were observed by inverted fluorescence microscope.Recombinant probe was constructed successfully and expressed efficiently in vitro.The in vitro assay demonstrated that the probe had greatly changed the fluorescence intensity ratio of FRET after adding Zn2+. When added excessive zinc ions, the probe decreased significantly at 475nm, increased greatly at 527nm, and the dynamic range of Rmax/Rmin is 3.0, which is very high.The probe also had high specificity towards Zn2+. While the other metal ions can cause changes in FRET to a certain extent, concentrations of these heavy metals in the living cells are very low, which will not significantly affect the detection of zinc ions in the living cells. The probe also had good responses to Zn2+ in vivo. From the imaging experiment of the probe in the living HeLa cells, we found out that the fluorescent intensity ratio changed greatly after adding zinc ions. Finally, we can calculate the concentration of free zinc ions in cells through the probe. |
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