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dc.contributor.authorWilliamson, Adele Kim
dc.contributor.authorPedersen, Hege
dc.date.accessioned2016-03-15T14:20:16Z
dc.date.available2016-03-15T14:20:16Z
dc.date.issued2014-05
dc.description.abstractThe 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.en_US
dc.descriptionAccepted manuscript version. Published version at <a href=http://doi.org/10.1016/j.pep.2014.02.008>http://doi.org/10.1016/j.pep.2014.02.008</a>. License in accordance with the journal's policy - <a href=http://creativecommons.org/licenses/by-nc-nd/4.0/>CC-BY-NC-ND</a>. http://dx.doi.org/10.1016/j.pep.2014.02.008en_US
dc.identifier.citationProtein Expression and Purification 2014, 97:29-36en_US
dc.identifier.cristinIDFRIDAID 1176619
dc.identifier.doi10.1016/j.pep.2014.02.008
dc.identifier.issn1096-0279
dc.identifier.urihttps://hdl.handle.net/10037/8968
dc.identifier.urnURN:NBN:no-uit_munin_8535
dc.language.isoengen_US
dc.publisherElsevieren_US
dc.relation.projectIDNorges forskningsråd: 192123
dc.rights.accessRightsopenAccess
dc.subjectAliivibrio salmonicidaen_US
dc.subjectMaltose-binding proteinen_US
dc.subjectRecombinant expressionen_US
dc.subjectATP-dependent DNA ligaseen_US
dc.subjectPeriplasmic localization sequenceen_US
dc.subjectVDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Bioinformatikk: 475en_US
dc.titleRecombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicidaen_US
dc.typeJournal articleen_US
dc.typeTidsskriftartikkelen_US
dc.typePeer revieweden_US


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