Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida

Protein Expr Purif. 2014 May;97:29-36. doi: 10.1016/j.pep.2014.02.008. Epub 2014 Feb 25.

Abstract

The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

Keywords: ATP-dependent DNA ligase; Aliivibrio salmonicida; Maltose-binding protein; Periplasmic localization sequence; Recombinant expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aliivibrio salmonicida / enzymology*
  • Aliivibrio salmonicida / genetics
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA Ligase ATP
  • DNA Ligases / chemistry
  • DNA Ligases / genetics*
  • DNA Ligases / isolation & purification
  • DNA Ligases / metabolism
  • Escherichia coli / genetics
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / genetics
  • Glutathione Transferase / isolation & purification
  • Glutathione Transferase / metabolism
  • Maltose-Binding Proteins / chemistry
  • Maltose-Binding Proteins / genetics
  • Maltose-Binding Proteins / isolation & purification
  • Maltose-Binding Proteins / metabolism
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Solubility

Substances

  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • DNA Ligases
  • DNA Ligase ATP