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

Abstract

The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATPdependent 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 E. 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-stransferase (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 Aliivibrio salmonicida ATPdependent DNA ligase. Highlights A putative periplasmic DNA ligase from Aliivibrio salmonicida was recombinantly produced in E. coli  Both the full-length and N-terminally truncated variants were expressed and purified  Toxicity of gene products to the host cells was overcame by fusion to large solubility tags  The truncated protein is more active in DNA ligation than the full-length version

Analysis of the distribution and evolution of the ATP‐dependent DNA ligases of bacteria delineates a distinct phylogenetic group ‘Lig E’

“…These enzymes have only the AD and OB domains, and all have a predicted N‐terminal signal sequence that would direct the mature protein to the periplasmic space of the bacterium, concomitant with its proteolytic removal. Although no in vivo experiments have been conducted to confirm an extracellular location, studies with recombinant b‐ADL from the fish pathogen Aliivibrio salmonicida show the leader‐less truncation variant of the protein is more stable and active than the full length construct (Williamson and Pedersen, ).…”

“…The present study investigates the structural organization of b‐ADLs and their distribution and diversity among bacterial taxa. Our analysis draws on the wealth of sequence information that has become available in recent years: a large number of bacterial genome sequences are now available, many b‐ADLs have been biochemically characterized (Weller et al ., ; Gong et al ., ; Magnet and Blanchard, ; Zhu and Shuman, 2005; 2007; de Vega, ; Williamson and Pedersen, ; Williamson et al ., ) and the structures of two b‐ADLs have been determined to high resolution (Akey et al ., ; Williamson et al ., ). We have first identified b‐ADLs in GenBank and classified them based on their domain arrangements.…”

Temperature adaptation of DNA ligases from psychrophilic organisms

“…All Lig E-type ADLs have strong predictions for N-terminal leader sequences proposed to direct them to the periplasm. Proposed biological functions of such secreted ligases include competence and DNA uptake in the periplasm (Magnet and Blanchard 2004), and the demonstrated increase in activity and solubility when this predicted leader was not included in recombinantly-produced Aliivibrio salmonicida (hereafter referred to as Vib-Lig) supports such signal processing (Williamson and Pedersen 2014).…”

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

“…To increase protein yields to quantities suitable for biophysical characterization, ParI was produced in fusion to an N-terminal His-tag and maltose binding protein (His-MBP), the latter of which is known to promote solubility (Fig. 3B) [46,47]. The MBP fusion partner was successfully removed by treatment with tobacco etch virus (TEV) protease (Fig.…”