Purification, crystallization and data collection of the apoptotic nuclease endonuclease G.
Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving chromosomal DNA. EndoG is the main apoptotic endonuclease in the caspase-independent pathway. Mouse EndoG without the mitochondrial localization signal (amino-acid residues 1-43) was successfully overexpressed, purified and crystallized using a microbatch method under oil. The initial crystal (type I) was grown in the presence of the detergent CTAB and diffracted to 2.8 A resolution, with unit-cell parameters a = 72.20, b = 81.88, c = 88.66 A, beta = 97.59 degrees in a monoclinic space group. The crystal contained two monomers in the asymmetric unit, with a predicted solvent content of 46.6%. Subsequent mutation of Cys110 improved the stability of the protein significantly and produced further crystals of types II, III and IV with space groups C2, P4(1)2(1)2 (or P4(3)2(1)2) and P2(1)2(1)2(1), respectively, in various conditions. This suggests the critical involvement of this conserved cysteine residue in the crystallization process.
Yoon
Sei Mee
SM
Medical Proteomic Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Song
Hyung Nam
HN
Yang
Jun Hyuk
JH
Lim
Mi Yeon
MY
Chung
Yong Je
YJ
Ryu
Seong Eon
SE
Woo
Eui Jeon
EJ
D000818:Animals; D017209:Apoptosis; D003460:Crystallization; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice
D004706:Endodeoxyribonucleases; C052391:endonuclease G