Cloning, purification and metal binding of the HNH motif from colicin E7

B. Gyurcsik, Anikó Czene, Hajnalka Jankovics, Noémi I. Jakab-Simon, Krystyna Ślaska-Kiss, A. Kiss, Z. Kele

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The HNH family of endonucleases is characterized by a ββα metal-finger structural motif. Colicin E7 is a representative member of this family containing the strictly conserved HNH motif at its C-terminus. Structural and biochemical studies suggested that the HNH motif could contain all the residues necessary for metal ion binding and nuclease activity. In this work a 43 amino acid peptide extending from V534 to K576 of colicin E7 and encompassing the HNH motif was cloned and expressed in Escherichia coli as a ubiquitin fusion protein. The N-terminal fusion tag was cleaved off by a specific protease, and the HNH peptide was purified free of ubiquitin. Circular dichroism, fluorescence and mass spectrometry showed that the zinc-ion binding affinity of the purified HNH peptide was much weaker than that of the intact nuclease domain suggesting that the N-terminal part of the nuclease domain is essential for stabilizing the structure of the HNH motif. The coordination sphere of the metal ion was found to be not fully equipped by the ligand - leaving a free coordination site for the substrate. Neither DNA binding nor DNAse activity of the purified HNH peptide was detected. Comparison of the glutathion-S-transferase-fused N-terminal deletion mutants of the colicin E7 nuclease domain suggested that the presence of the DNA-binding site is still not sufficient for the catalytic activity.

Original languageEnglish
Pages (from-to)210-218
Number of pages9
JournalProtein Expression and Purification
Volume89
Issue number2
DOIs
Publication statusPublished - 2013

Fingerprint

Colicins
Organism Cloning
Metals
Peptides
Ions
Ubiquitin
Endonucleases
DNA
Fluorescence Spectrometry
Circular Dichroism
Glutathione Transferase
Fingers
Zinc
Mass Spectrometry
Peptide Hydrolases
Binding Sites
Escherichia coli
Ligands
Amino Acids
Proteins

Keywords

  • Circular dichroism
  • Gel mobility shift
  • HNH nuclease
  • Mass spectrometry
  • Ubiquitin fusion
  • Zn-binding

ASJC Scopus subject areas

  • Biotechnology

Cite this

Cloning, purification and metal binding of the HNH motif from colicin E7. / Gyurcsik, B.; Czene, Anikó; Jankovics, Hajnalka; Jakab-Simon, Noémi I.; Ślaska-Kiss, Krystyna; Kiss, A.; Kele, Z.

In: Protein Expression and Purification, Vol. 89, No. 2, 2013, p. 210-218.

Research output: Contribution to journalArticle

Gyurcsik, B. ; Czene, Anikó ; Jankovics, Hajnalka ; Jakab-Simon, Noémi I. ; Ślaska-Kiss, Krystyna ; Kiss, A. ; Kele, Z. / Cloning, purification and metal binding of the HNH motif from colicin E7. In: Protein Expression and Purification. 2013 ; Vol. 89, No. 2. pp. 210-218.
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