Expression,Purification And Crystallization Of SEY1p Protein In Candida Albicans

Dynamin GTPases superfamily,which catalyzes GTP hydrolysis,has many members that are involved in multiple cellular events which include fission and fusion of organelles,endocytosis,vesicle scission and cytokinesis.Although they function in many cellular scenarios,they have a similar architecture comprising the N-terminal G domain that participates in nucleotide binding and hydrolysis,a middle domain and a GTPase effector domain that regulates and responds to hydrolysis of GTPase domain.SEY1p,a member of dynamin GTPase superfamily,localizes to the endoplasmic reticulum in S.cerevisiae.It has an N-terminal GTPase domain,a predicted helical bundle,two closely spaced transmembrane segments and a C-terminal tail.The deletion of SEY1 p results in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified SEY1 p fuse in a GTP-dependent manner in vitro.These results indicate that SEY1 p mediates homotypic ER fusion,but the molecular mechanism how the SEY1 p mediates homotypic membranes fusion is unclear.In this study,E.coli expression system was used to express SEY1 p except the transmembrane domain.The target protein was purified by Ni-NTA affinity chromatography and ion-exchange chromatography.Then crystals were obtained using the hanging-drop vapor-diffusion technique.Finally,we collected a set of data of SEY1 p by the method of X-ray diffraction at 2.9 ? resolution and laid the foundation for elucidating how SEY1 p mediates the homotypic membranes fusion.