Biochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex.
Biochimie
; 88(3-4): 245-51, 2006.
Article
in En
| MEDLINE
| ID: mdl-16376476
ABSTRACT
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 11 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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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Pseudomonas aeruginosa
/
Bacterial Proteins
/
Serine Endopeptidases
/
Alginates
Type of study:
Prognostic_studies
Language:
En
Journal:
Biochimie
Year:
2006
Type:
Article
Affiliation country:
New Zealand