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Profiling and comprehensive expression analysis of ABC transporter solute‐binding proteins of Bacillus subtilis membrane based on a proteomic approach
Author(s) -
Bunai Keigo,
Ariga Masanori,
Inoue Taro,
Nozaki Manabu,
Ogane Shinya,
Kakeshita Hiroshi,
Nemoto Tadashi,
Nakanishi Hiroshi,
Yamane Kunio
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305676
Subject(s) - membrane protein , biochemistry , chemistry , gel electrophoresis , proteome , bacillus subtilis , amino acid , biology , membrane , chromatography , microbiology and biotechnology , bacteria , genetics
Abstract We analyzed ABC transporter solute‐binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 m M nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) or two one‐dimensional (1‐D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2‐D gel separation profiles of submembrane fractions solubilized by 1% n ‐dodecyl‐β‐ D ‐maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn ++ , glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).