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Protein Protein Interactions

4.1 Protein-protein interactions revealed by in vitro and in vivo pull-down assay

For examination of direct interaction between Ter proteins of interest, we employed the pull-down assay. This in vitro method is a useful tool for determination of a protein-protein interaction. Successful utilization of this assay requires previously optimized and handled purification of tagged protein (the bait) which is crucial for capturing its binding partner (the prey). Full-length essential tellurite resistance genes encoding TerB, TerC, TerD and TerE were cloned into several expression vectors as described in Materials and Methods. All possible combinations of interacting Ter protein pairs were examined using in vitro pull down assay (Table PDA1, Figure PDW2ab). Both bait and prey protein carried a tag fused to their N termini. The His-Ter bait protein, immobilized on Ni-NTA affinity matrix, was intended to capture GST-tagged Ter prey protein. After several washes, the presence of retained proteins was detected by Western blot using antibodies against His-tag, as well as anti-GST antibodies (Figure PDW2ab). The homotypic interactions were detected for TerB, TerD, and TerE proteins. We also detected the heterotypic interactions between TerB-TerC, TerB-TerD, TerB-TerE, TerD-TerE in both combinations of prey and target plasmids. The strong interactions were discriminated for the pair TerD-TerE (Figure PDW2b).

Table PDA1:

Bait expression plasmid

Bait Protein , molecular weight

Prey expression plasmid

Prey Protein , molecular weight

Figure

pETterB

His-TerB, ~20.25 kDa

pGEXterB

GST-TerB, ~42.98 kDa

Figure 7657

pETterB

His-TerB, ~20.25 kDa

pGEXterC

GST-TerC, ~64.68 kDa

Figure 7660

pRSFDuet(terB)

His-TerB, ~20.25 kDa

pGEXterD

GST-TerD, ~46.78 kDa

Figure 7635

pRSFDuet(terB)

His-TerB, ~20.25 kDa

pGEXterE

GST-TerE, ~46.76 kDa

Figure 7636

pETterC

His-TerC, ~41.95 kDa

pGEXterB

GST-TerB, ~42.98 kDa

Figure 7656

pETterC

His-TerC, ~41.95 kDa

pGEXterC

GST-TerC, ~64.68 kDa

Figure 7659

pETterC

His-TerC, ~41.95 kDa

pGEXterD

GST-TerD, ~46.78 kDa

Figure 7630

pETterC

His-TerC, ~41.95 kDa

pGEXterE

GST-TerE, ~46.76 kDa

Figure 7631

pETterD

His-TerD, ~24.12 kDa

pGEXterC

GST-TerC, ~64.68 kDa

Figure 7655

pETterD

His-TerD, ~24.12 kDa

pGEXterD

GST-TerD, ~46.78 kDa

Figure 7634

pETterD

His-TerD, ~24.12 kDa

pGEXterE

GST-TerE, ~46.76 kDa

Figure 7628

pETterE

His-TerE, ~24.03 kDa

pGEXterB

GST-TerB, ~42.98 kDa

Figure 7654

pETterE

His-TerE, ~24.03 kDa

pGEXterC

GST-TerC, ~64.68 kDa

Figure 7658

pETterE

His-TerE, ~24.03 kDa

pGEXterD

GST-TerD, ~46.78 kDa

Figure 7624

pETterE

His-TerE, ~24.03 kDa

pGEXterE

GST-TerE, ~46.76 kDa

Figure 7627

4.1.1 invivo pull-down assay

Because there was the possibility that multimeric aggregates (oligomeric structures) of proteins could not interact in vitro and limit to bind onto the Ni-NTA resin. Therefore, we selected the Duet-coexpression system with the pGEX-4T-1 system to coexpress two proteins, allowing possible interaction inside the cells and consequently purifying the protein complex. In this case, one protein carries the polyhistidine tag and could be affinity purified on a Ni column and the second was produced as a GST-fused protein that could be pulled down. Immunoblot analysis of proteins extracts using antibodies to His-tag or GST-tag confirmed that all studied Ter proteins were produced in E. coli BL21(DE3). To identify potential interactions, the proteins after purification on Ni agarose were analyzed by Western blotting. The results obtained from coexpression experiment (Figure 2cd) confirmed that they are in accordance with in vitro pull down generated outputs. Both results suggest that TerD protein made direct strong contact with its TerE protein partners.

To overcome the possibility that in the negative cases (TerC dimerisation or interaction of TerC with TerD/E) large protein complexes or oligomeric structures could not interact in vitro or limit binding to Ni-NTA resin we decided to perform the in vivo co-expression of the proteins by the compatible Duet and pGEX-4T-1 systems. After simultaneous overproduction and purification on Ni agarose the samples were again analyzed by Western blotting. The results obtained from co-expression experiment (Figure 1cd) confirmed in vitro pull-down results. Two bands visible on western blot suggest the strong heterodimerisation of TerE and TerD proteins.

Figure PDW2: Western blot of protein interactions among the tellurite resistance proteins. Recombinant Ter proteins were probed with monoclonal anti-His-tag antibody (a, c) and monoclonal anti-GST-tag antibody (b, d). Above each lane is the description of samples used for protein-protein interaction study. Molecular weight standard (kDa) is indicated on the left side of each panel. Panels a and b show the results of in vitro pull-down assay. His-Ter proteins were expressed in E. coli BL21(DE3) transformed by bait vector (marked BP). Prey proteins (GST-Ter) were produced in E. coli BL21(DE3) with prey vector (marked PP). Lanes represent the eluted proteins probed by corresponding antibodies. Panels c and d show the elution profiles of co-expression analyses. Bait and prey proteins were co-expressed together in the same cell.

4.1.1 Separation of TerD/TerE protein dimmers by native PAGE electrophoresis

To visualize the protein association we further used the elution extract after in vivo co-expression and Ni-agarose binding of His-TerE and GST-TerD pair. At first we separated the extract on the native PAGE (Figure NPDE2a) and then on the denaturing SDS PAGE (Figure NPDE2b). Several distinct bands could be identified and could be assigned according the molecular weight to either single proteins or hetero- or homodimers (Figure NPDE2b). Unfortunately this approach was unsuccessful with co-expression of all four Ter proteins because of low separation ability of native PAGE gel. This finding indicated that the high molecular species which probably formed could not enter the native PAGE gel. However, when the Ni-agarose extract was isolated by non-denaturing method and then separated on SDS-PAGE bands with sizes higher then the single expressed proteins. The sizes of band can be appointed to predicted sizes of various protein assemblages (Figure NPDE2c). Importantly, when the proteins were isolated by urea denaturing method and then separared by SDS-PAGE these bands disappeared (Figure NPDE2d). These observation together further confirm association of the Ter proteins.

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