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dc.contributor.authorGutsche, J
dc.contributor.authorRemminghorst, U
dc.contributor.authorRehm, BHA
dc.date.accessioned2018-03-13T03:03:40Z
dc.date.available2018-03-13T03:03:40Z
dc.date.issued2006
dc.identifier.issn0300-9084
dc.identifier.doi10.1016/j.biochi.2005.06.003
dc.identifier.urihttp://hdl.handle.net/10072/371079
dc.description.abstractAlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein–protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.
dc.description.peerreviewedYes
dc.languageEnglish
dc.language.isoeng
dc.publisherElsevier
dc.relation.ispartofpagefrom245
dc.relation.ispartofpageto251
dc.relation.ispartofissue3-4
dc.relation.ispartofjournalBiochimie
dc.relation.ispartofvolume88
dc.subject.fieldofresearchBiochemistry and cell biology
dc.subject.fieldofresearchBiochemistry and cell biology not elsewhere classified
dc.subject.fieldofresearchcode3101
dc.subject.fieldofresearchcode310199
dc.titleBiochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.hasfulltextNo Full Text
gro.griffith.authorRehm, Bernd


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