Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans

Jana Kieper, Christiane Lauber, Oleg Gimadutdinow, Anna Urbaǹska, Iwona Cymerman, Mahua Ghosh, Bartosz Szczesny, Gregor Meiss

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'- exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities.

Original languageEnglish (US)
Pages (from-to)99-106
Number of pages8
JournalProtein Expression and Purification
Volume73
Issue number1
DOIs
StatePublished - 2010

Fingerprint

Recombinant Proteins
Yeasts
Endonucleases
Escherichia coli
Substrate Specificity
DNA Repair
Genetic Recombination
Cell Death
Enzymes
Exonucleases
Inclusion Bodies
Caenorhabditis elegans
Caspases
Mitochondrial DNA
Cell Biology
Organism Cloning
Apoptosis
endonuclease G
Proteins

Keywords

  • Apoptosis
  • DNA-repair
  • Endonuclease
  • Exonuclease
  • Mitochondria
  • Refolding

ASJC Scopus subject areas

  • Biotechnology

Cite this

Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans. / Kieper, Jana; Lauber, Christiane; Gimadutdinow, Oleg; Urbaǹska, Anna; Cymerman, Iwona; Ghosh, Mahua; Szczesny, Bartosz; Meiss, Gregor.

In: Protein Expression and Purification, Vol. 73, No. 1, 2010, p. 99-106.

Research output: Contribution to journalArticle

Kieper, Jana ; Lauber, Christiane ; Gimadutdinow, Oleg ; Urbaǹska, Anna ; Cymerman, Iwona ; Ghosh, Mahua ; Szczesny, Bartosz ; Meiss, Gregor. / Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans. In: Protein Expression and Purification. 2010 ; Vol. 73, No. 1. pp. 99-106.
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