Screening system to detect Trx-TF interaction in <i>E</i>. <i>coli</i> cells.

<p>(<b>A</b>) Schematic representation of the <i>E</i>. <i>coli</i> co-expression screening system. The S-tagged mutant TrxM, whose active site C35 was substituted with serine and a His-tagged TF, are co-expressed in <i>E</i>. <i>coli</i> (Origami2 strain) cells. The Trx-TF complex can be detected by immunoblot analysis using either S-protein or a His-tag antibody. (<b>B</b>) Expression levels of PedR and Trx in the control Origami2 strain (Control), the strain expressing only TrxM_C35S (Trx), the strain expressing only PedR (PedR) and the strain expressing both TrxM_C35S and PedR (Trx-PedR). The whole cell extract (C), the soluble fraction (S) and the insoluble pellet fraction (P) were separated by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (<b>C</b>) Detection of the interaction between PedR and Trx. After the <i>E</i>. <i>coli</i> soluble protein fraction was separated by non-reducing SDS-PAGE, PedR and Trx were detected by immunoblot analysis using a His-tag antibody (left) and S-protein (right), respectively. ± indicates with or without 100 mM DTT treatment. Black arrow, white arrow and arrow head indicate the PedR monomer, the Trx monomer, and the Trx-PedR complex, respectively.