Zhou Yangbin, Song Jiaping, Weng Linhong, Hu Xiaojian, Ding Yu, Zhang Zhihong
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Protein Pept Lett. 2007;14(9):928-32. doi: 10.2174/092986607782110338.
The technique of fluorescence (or Förster) resonance energy transfer (FRET) is widely used to observe bimolecular interaction in living cells. Cyan and yellow fluorescent proteins are the most widely used pair in FRET analysis. CyPet and YPet are two newly optimized fluorescent proteins that have much better dynamic range and sensitivity than CFP/YFP pair, although the crystallographic structure and the mechanism of better fluorescent characteristics of CyPet are still unknown. We have expressed the cyan fluorescent protein CyPet using pT7 prokaryocyte expression system in Escherichia coli strain Rosetta (DE3) pLysS by auto-induction. After purification, the recombinant CyPet protein was crystallized by hanging drop vapor diffusion technique and could diffract to 2.55A resolution. The data showed that the orthorhombic CyPet crystal was in space group P212121 with unit cell parameters (51.55, 61.53, 63.36) and contained one molecule in one asymmetric unit.
荧光（或Förster）共振能量转移（FRET）技术被广泛用于观察活细胞中的双分子相互作用。青色和黄色荧光蛋白是FRET分析中使用最广泛的一对。CyPet和YPet是两种新优化的荧光蛋白，其动态范围和灵敏度比CFP/YFP对要好得多，尽管CyPet的晶体结构和更好荧光特性的机制仍不清楚。我们通过自动诱导，使用pT7原核细胞表达系统在大肠杆菌Rosetta (DE3) pLysS菌株中表达了青色荧光蛋白CyPet。纯化后，通过悬滴气相扩散技术使重组CyPet蛋白结晶，并能衍射到2.55埃的分辨率。数据显示，正交晶系的CyPet晶体属于空间群P212121，晶胞参数为(51.55, 61.53, 63.36)，一个不对称单元中包含一个分子。
