Journal: Biochemistry and Biophysics Reports

Article Title: A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

doi: 10.1016/j.bbrep.2016.05.010

Figure Lengend Snippet: SDS-PAGE gel profile of proteins eluted from the His-MamA column and their apparent molecular masses; lane M, protein markers (Precision Plus protein standards; Bio-Rad); lane 1, eluted proteins. The eluted fractions were concentrated approximately 200 times for SDS-PAGE. These 13 bands were analyzed using tandem mass spectrometry and identified. Bands 2, 4, and 6 were proteins belonging to E. coli ; bands 1, 3, 5, and 12 were proteins belonging to M. magneticum AMB-1; and bands 7–11, and 13 were recombinant MamA proteins. Only two of the bands were identified as magnetosome associated proteins, Mms6 and MamA. The gel was stained with Coomassie Brilliant Blue G-250.

Article Snippet: For C-terminal His-tagged full-length Mms6 expression, the plasmid pET29b-mms6 1–133 was constructed by cloning the entire PCR-amplified mms6 gene ( mms6 1–133 ; accession number: AB096081) fragment into the Nde I /Kpn I sites of pET-29b (Merck-Millipore) using a primer set (mms6–1f and mms6–1r).

Techniques: SDS Page, Mass Spectrometry, Recombinant, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

doi: 10.1016/j.bbrep.2016.05.010

Figure Lengend Snippet: (A) Immunoblotting of M. magneticum AMB-1 extracts labeled with anti-Mms6 1–133 polyclonal antibodies [left]. Two different Mms6 bands are evident, one at 14.5-kDa (arrow) and the other at 6.0-kDa (arrowhead). The 14.5-kDa Mms6 has a higher intensity than the 6.0-kDa Mms6. In the control experiment, the immunoblotting was carried out with an excess amount of Mms6 1–133 antigen. In the control, the 14.5-kDa and 6.0-kDa bands were not detected [right]. S: soluble fraction; M: membrane fraction; MA: magnetosome fraction. (B) Two methods were used to extract Mms6 from the magnetosomes and then analyzed using immunoblotting. Method I used 2% SDS at 37 °C for 1 h to extract Mms6 which produced two Mms6 bands, one at 14.5-kDa and another at 6.0-kDa, but the 14.5-kDa band has higher intensity. Method II was performed by Arakaki et al. which extracted Mms6 by boiling magnetosomes in 1% SDS for 1.5 h with three aliquots taken every 30 min lane 1, 2, and 3. This resulted in two distinct Mms6 bands (14.5-kDa [arrow] and 6.0-kDa [arrowhead]) in the first aliquot but only one band (14.5-kDa) in the second two aliquots.

Article Snippet: For C-terminal His-tagged full-length Mms6 expression, the plasmid pET29b-mms6 1–133 was constructed by cloning the entire PCR-amplified mms6 gene ( mms6 1–133 ; accession number: AB096081) fragment into the Nde I /Kpn I sites of pET-29b (Merck-Millipore) using a primer set (mms6–1f and mms6–1r).

Techniques: Western Blot, Labeling, Produced

Journal: Biochemistry and Biophysics Reports

Article Title: A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

doi: 10.1016/j.bbrep.2016.05.010

Figure Lengend Snippet: (A) SDS-PAGE analyses of the immunoprecipitation assays. A mixture containing His-MamA and Mms6 1–133 -His was precipitated with anti -MamA (left) or anti-Mms6 1–133 (right) antibodies and clearly show that Mms6 (arrow) co-precipitates with MamA (arrowhead). When normal serum was used, there was no band for either MamA or Mms6 (right lanes). (B) SDS-PAGE analyses of the Ni-NTA agarose pull-down assay. The arrows indicated the His-MamA and the His-tag removed MamA protein bands; the arrowheads indicated the Mms6 1–133 -His and His-tag removed Mms6 1–133 protein bands. Both the immunoprecipitation and pull-down assays confirm the interaction between MamA and Mms6 1–133 . The molecular mass standards (Precision Plus protein standards; Bio-Rad) are indicated on the left side of the gels. The gels were stained with Coomassie Brilliant Blue G-250.

Article Snippet: For C-terminal His-tagged full-length Mms6 expression, the plasmid pET29b-mms6 1–133 was constructed by cloning the entire PCR-amplified mms6 gene ( mms6 1–133 ; accession number: AB096081) fragment into the Nde I /Kpn I sites of pET-29b (Merck-Millipore) using a primer set (mms6–1f and mms6–1r).

Techniques: SDS Page, Immunoprecipitation, Pull Down Assay, Staining