Expression, purification, crystallization and preliminary x-ray analysis of Cyan fluorescent protein CyPet.
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.
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Yangbin
Y
Zhou
Jiaping
J
Song
Linhong
L
Weng
Xiaojian
X
Hu
Yu
Y
Ding
Zhihong
Z
Zhang
D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
C485182:Cyan Fluorescent Protein; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins