The pSUMO-based SUMOylation system modifies proteins in <i>E.coli</i>.

<p>(<b>A</b>) Scheme of the <i>in vivo</i> SUMO maturation, SUMO conjugation and deconjugation process (for detailed description, see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102157#s1" target="_blank">Introduction</a>”). (<b>B</b>) pSUMO vectors containing the humanized SUMOylation system consisting of N-terminally His₆-tagged SUMO1, 2 or 3, the SUMO-conjugating enzyme E2 (Ubc9) and both subunits of the SUMO-activating enzyme (SAE1 and SAE2) as a cistronic expression unit with an internal ribosomal binding site (rbs). Expression of the respective cDNAs is under the control of a lac-repressor (LacI) regulated T7 promoter. (<b>C</b>) Scheme of the experimental setup of the pSUMO-based in-cell SUMO conjugation. <i>E.coli</i> BL21 cells were used containing pSUMO1 in combination with pTG-mTDG or pGEX-hXRCC1 plasmids were used for the co-expression of the complete SUMO system with C- and N-terminally GST-tagged mTDG and hXRCC1, respectively. Immunoblot analyses of mTDG (<b>D</b>) and hXRCC1 (<b>E</b>) SUMOylation in <i>E.coli</i> cells, expressing the SUMO target and the SUMO system from either the pSUMO1 or the pT-E1E2S1 plasmid (250 µM IPTG at 25°C for 2 h). Co-expression of target proteins mutated in the SUMO acceptor sites of mTDG (K341R) and hXRCC1 (K176R) were included to assess the specificity of the SUMOylation system.