Biochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex
Author(s)
Gutsche, J
Remminghorst, U
Rehm, BHA
Griffith University Author(s)
Year published
2006
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AlgX 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 ...
View more >AlgX 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.
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View more >AlgX 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.
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Journal Title
Biochimie
Volume
88
Issue
3-4
Subject
Biochemistry and cell biology
Biochemistry and cell biology not elsewhere classified