Expression, purification, crystallization and preliminary X-ray analysis of cyan fluorescent protein CyPet

The technique of fluorescence (or Forster) 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 2.55 angstrom 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.