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. 2015 Mar 16;10(3):e0119761. doi: 10.1371/journal.pone.0119761

Fig 7. TatA/Tat substrate interactions in solution.

Fig 7

(A) Soluble non-recombinant TatA co-purifies with the precursor of HiPIP and NrfC and not with their mature forms. Precursor and mature forms of HiPIP were produced in E. coli strain MC4100 using pEXH5tac-H6 and pEXH5tac-mat-H6, respectively. The MalE-signal peptide-HiPIP fusion protein was produced using pEXH5tac-malE(sp)-H6, and the RR>KK mutated HiPIP variant was produced using pEXH5tac-H6-KK. Precursor and mature forms of NrfC were produced using pBW-nrfC-H6, pBW-nrfC-mat-H6, pBW-nrfC-strep or pBW-nrfC-mat-strep, as indicated. Soluble protein (S), flow-through (F), wash (W) and elution fractions (E1-6) were analyzed by SDS-PAGE Western blotting, using antibodies directed against HiPIP, the H6-Tag (in case of NrfC), or TatA as indicated. (B) In vitro folded pure HiPIP precursor associates with tagged soluble TatA. 5 μM of precursor (left panel) or mature (right panel) HiPIP were added to crude soluble extracts containing wild-type level TatA-strep (from strain DADE tatA-strep-tatBC), incubated at room temperature for 20 min and used for affinity chromatography. Further analyses were carried out as in A), using antibodies directed against TatA or HiPIP, as indicated. Arrows in A) and B) indicate co-elutions. p, precursor; m, mature form; BCCP, biotin carboxyl carrier protein.