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. 2016 May 24;7(3):e00427-16. doi: 10.1128/mBio.00427-16

FIG 3 .

FIG 3 

Examination of AcsA translation pattern with FLAG-tagged protein in C. autoethanogenum and E. coli. (A) Western blot analysis of C. autoethanogenum transconjugant crude lysates. Lanes: M, Bio-Rad kaleidoscope Precision Plus protein ladder; 1, 13 µg of soluble lysate of pMTL8315-PacsA plasmid control; 2, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TGA)-FLAG; 3, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TCA)-FLAG; 4, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TAA)-FLAG; 5, 6.7 µg of insoluble lysate of pMTL83151-PacsA plasmid control; 6, 6.7 µg of insoluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA). The red arrow indicates the position of a mature 69-kDa protein; the blue arrow indicates the position of the larger 44-kDa truncated protein. The ca. 70-kDa band present in all crude lysates from C. autoethanogenum is a consequence of nonspecific binding of the anti-FLAG antibody to a native C. autoethanogenum protein, most likely DnaK (encoded by CAETHG_2891), which shares 7/8 amino acid identity with FLAG and is of the appropriate predicted size. (B) Schematic showing the expected protein sizes of C. autoethanogenum AcsA in the event of translational readthrough or truncation. (C) Western blot analysis of E. coli transformant crude lysates. Lanes: M, Bio-Rad kaleidoscope Precision Plus protein ladder; 7, 15 µg of soluble lysate of pMTL83151-PacsA plasmid control; 8, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TGA)-FLAG; 9, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TCA)-FLAG; 10, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TAA)-FLAG; 11, 39 µg of soluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA); 12, 38 µg of insoluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA).