Figure 1.
The pSUMO-based SUMOylation system modifies proteins in E.coli.
(A) Scheme of the in vivo SUMO maturation, SUMO conjugation and deconjugation process (for detailed description, see “Introduction”). (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. (C) Scheme of the experimental setup of the pSUMO-based in-cell SUMO conjugation. E.coli 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 (D) and hXRCC1 (E) SUMOylation in E.coli 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.
Figure 2.
Purification of SUMO1-modified mTDG and hXRCC1 produced by in-cell SUMOylation.
(A) Purification scheme for in-cell SUMO-modified protein. Cell lysates are subjected to subsequent GST- and Ni-NTA-affinity purification (work flow 1) or vice versa (work flow 2). Boxed letters indicate the corresponding sub-panels. Fractions of the purification of SUMO1-modified mTDG from purification work flow 1 (B) and 2 (D) and hXRCC1 from work flow 1 (F) were analyzed by SDS-PAGE and subsequent Coomassie blue staining and immunoblotting using monoclonal anti-GMP1 (C), polyclonal anti-mTDG (E) and anti-hXRCC1 (G) antibodies, respectively. in, input (cleared lysate or dialyzed elution fractions); f, flow through; w, wash steps; e, elution fractions; *, SUMO1-modified truncated mTDG.
Figure 3.
In-cell SUMOylation of TDG with the SUMO-E2-fusion system.
Scheme of the SUMO-activating vectors pSA1-3, which are identical to the pSUMO1-3 vectors but lack the Ubc9 expression unit (A) and the Ubc9-fusion SUMO-conjugating vectors pSC-PreE2 (E) and pSC-IntE2 (C) for expression of target protein fused to a GST-tagged Ubc9 under the control of the T7 promoter. A PreScission protease cleavage site or a Mycobacterium xenopi GyrA intein sequence in the linker region allows for the release of the modified target from the Ubc9-GST fusion. (B) Experimental setup of in-cell SUMO conjugation with the SUMO-E2-fusion system. pSA1 is co-expressed with target proteins either from pSC-IntE2 or pSC-PreE2 vectors in E.coli BL21. Boxed letters indicate the corresponding sub-panels. Immunoblot analysis of lysates of E.coli cells expressing the SUMO-activating proteins (pSA1) and wild-type (wt) or SUMO acceptor site-mutated (K330A) human TDG (hTDG) from the SUMO-conjugating vectors pSC-IntE2 (D) or pSC-PreE2 (F). Expression was induced with 250 µM IPTG at 15°C for 3 and 6 h or with 1 mM IPTG at 37°C for 1 h. (G) SUMOylation of mouse TDG (mTDG) expressed from the pSC-IntE2 vector was followed over time by immunoblot analysis. Expression was induced with 500 µM IPTG and cells were incubated at 20°C.
Figure 4.
The SUMO-E2-fusion system allows SUMOylation of targets in crude cell extracts.
(A) In-extract SUMOylation procedure using lysate from pSUMO1- or pSA1-expressing bacteria. Boxed letters indicate the corresponding sub-panels. In-extract SUMOylation efficiency with or without the addition ATP to a final concentration of 5 mM (30°C for 1 h) was assessed by immunoblot analysis. Extracts from E.coli BL21(DE3) cells expressing the SA1 (B) or the SUMO1 (C) system (250 µM IPTG at 30°C for 3 h) were mixed with extracts from cells expressing the fusion of Ubc9 to wild-type hXRCC1 (wt) or hXRCC1-K176R from the pSC-PreE2 plasmid (250 µM IPTG at 25°C for 3 h) with the indicated volume (V) ratio. (D) Direct comparison of the SUMOylation efficiency of hXRCC1-PreE2 and hXRCC1 not fused to Ubc9 with either the SA1 or SUMO1 extracts. (E) Crude E.coli BL21(DE3) cell extracts expressing wild-type (wt) or SUMO acceptor site-mutated hXRCC1 (K176R) (250 µM IPTG at 25°C for 3 h) from the pSC-IntE2 plasmids were mixed with extracts with the SA system (250 µM IPTG at 30°C for 3 h) at the indicated volume (V) ratio. Applying the same experimental conditions as above, the SUMOylation of wild-type (wt) and the SUMOylation-deficient (K330A) hTDG mutant was analyzed comparing co-incubation of extracts from E.coli BL21(DE3) cells expressing pSA1 and the TARGET-IntE2-fusion (F) or pSUMO1 and the non-Ubc9 fusion (G).
Figure 5.
In-cell SUMOylated TDG is active and shows enzymatic turnover.
200 ng of purified mouse TDG (mTDG), purified in-cell SUMOylated mTDG and in vitro SUMOylated samples were analyzed by immunoblot analysis using anti-mTDG (A) as well as anti-SUMO1 (B) antibodies. Conjugated SUMO1 was cleaved with the recombinant SUMO protease SenP2 at RT for 30 min (lanes 2, 4, 6) and compared to untreated samples (lanes 1, 3, 5). (C) Enzymatic activity and turnover of unmodified TDG (mTDG), in vitro (mTDG-SUMO1 i.v.) and purified in-cell SUMOylated TDG (mTDG-SUMO1) were assessed by the base release assay with a 10-fold molar excess of G·U mismatched oligonucleotides over enzyme. Samples were taken at the indicated time-points and the relative amounts of processed 23 nucleotide product (23 nt) versus unprocessed 60 nucleotide substrate (60 nt) was quantified and depicted in (D). Error bars, SEM of 2 experiments.
Figure 6.
Overview on our newly introduced SUMOylation systems indicating advantages, disadvantages and putative applications in comparison to host in vivo and purely in vitro systems.