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Purification, crystallization and data collection of the apoptotic nuclease endonuclease G.

Authors
  • Yoon, Sei Mee
  • Song, Hyung Nam
  • Yang, Jun Hyuk
  • Lim, Mi Yeon
  • Chung, Yong Je
  • Ryu, Seong Eon
  • Woo, Eui Jeon
Type
Published Article
Journal
Acta Crystallographica Section F Structural Biology and Crystallization Communications
Publisher
International Union of Crystallography
Publication Date
May 01, 2009
Volume
65
Issue
Pt 5
Pages
504–507
Identifiers
DOI: 10.1107/S1744309109013335
PMID: 19407388
Source
Medline
License
Unknown

Abstract

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.

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