Heterologous expression of NTD and CTD in E coli

To investigate in more detail the potential association between NTD and CTD of Pol5 revealed by our trans-complementation assay in vivo, we performed the heterologous expression and purification of Pol5 and both protein domains from E. coli cells. In the future, efficient purification of the domains from E. coli cells might also allow high- resolution structural analysis.

Sequences encoding the fragment genes pol5-ΔN677 (CTD) and pol5-Δ679L (NTD) were cloned in IPTG-inducible expression vectors and flanked at N- and C-termini by FLAG and HIS-tag coding sequences, respectively. C-terminally fused HIS tag was used for affinity purification with Ni-NTA agarose beads (see 4.2.1.7). To characterize the purification process, several fractions collected during the experiment were analyzed by western blotting and Coomassie staining (see 4.2.5) (Figure 55A to 55D).

To achieve efficient expression and purification of Pol5 domains as presented below, several conditions were tested at critical steps of the process. To optimize the expression process, several IPTG concentrations (between 0.05 and 0.5 M) for induction, different

time periods (between two and 18 hours), and temperatures (between 16°C and 37°C) for overexpression were tested. The most efficient expression was obtained by inducing the cells with 0.1 M IPTG and culturing for six hours at 24°C (see 4.2.1.6). To lyse the cells, sonication, as well as Precellys and vortexer in the presence of Zirconia-Silicate beads were tested. Best results with highest protein concentrations in the soluble fraction were obtained with vortexer and Zirconia-Silicate beads as described in detail in section 4.2.1.7. Moreover, different buffers were tried for cell lysis, purification, washing, and elution. The presented results were obtained using 20 mM sodium phosphate buffer (pH 7.8), 500 mM NaCl, and 20 mM imidazole for cell lysis and binding on Ni-NTA beads. Washing steps were performed with the same buffer containing 60 mM imidazole, and elution was more efficient with the same buffer but containing 250 mM imidazole (see also Table 9).

Figure 55: Recombinant purification of the CTD and NTD of Pol5 from E. coli.

(A, B) As indicated below on the left, the FLAG- and HIS-tagged CTD of Pol5 (referred to as ΔN677 or CTD) expressed in E. coli was purified with Ni-NTA coated agarose beads. Below on the right, blue bar depicts CTD with color-coded promoter (Prom.), FLAG tag, and HIS tag. 0.05% of whole cell lysates (WCL) and flow- through fractions (FT), 0.2% of wash steps (W1, W5, W10), and 2% (panel A) or 3% (panel B) of eluates and bead fractions (E1, E2, E3, BaE) were resolved on 10% SDS-PAGEs. Protein detection was performed using western blotting and an antibody against HIS (A) or Coomassie staining (B). (C, D) As indicated below on the left, the FLAG- and HIS-tagged NTD of Pol5 (referred to as Δ679L or NTD) expressed in E. coli was purified with Ni-NTA coated agarose beads. Below on the right, red bar depicts NTD with color-coded promoter (Prom.), FLAG tag, and HIS tag. For western blotting and detection with antibody against HIS (panel C), 0.1% of whole cell lysates (WCL) and flow-through fraction (FT), 0.2% of wash steps (W1, W5, W10), and 2% of eluates and bead fractions (E1, E2, E3, BaE) were resolved on 8% SDS-PAGE. For Coomassie staining (panel D), 0.2% of whole cell lysates (WCL), flow-through fraction (FT), and wash steps (W1, W5, W10), 6% of eluates 1 (E1), and 3% of remaining eluates and bead fractions (E2, E3, BaE) were resolved on 8% SDS-PAGE.

Regarding the purification of ΔN677, both western blot analysis with an antibody against HIS (Figure 55A) and Coomassie staining (Figure 55B) showed that very high amounts of the protein migrating at 55 kDa were expressed (see WCL) and purified (see E1 and E2). The smaller bands between 22 and 32 kDa likely resulted from C-terminal degradation during expression.

In contrast, the expression of the Δ679L polypeptide, with a molecular weight around 78 kDa, was less efficient in E. coli (see WCL). Moreover, the NTD was less stably bound to the beads than the CTD because high amounts of protein were detected in the first wash fraction (see W1). Unexpectedly, elution of this polypeptide was consistently less efficient compared to the CTD since comparatively high amounts of protein were retained on the beads (see E1, E2, and BaE). In addition, we observed a smaller band in all relevant fractions, which might correspond to a stable degradation product of Pol5-NTD from its N-terminus.

Figure 56: Recombinant co-purification of the NTD with the CTD of Pol5 from E. coli.

Below, colored bar depicts NTD of Pol5 (referred to as Δ679L or NTD; red) fused to FLAG tag at N-terminus and CTD of Pol5 (referred to as ΔN677 or CTD; blue) fused to HIS tag at C-terminus. Domains are separated by shine-dalgarno sequence (SD; mint) and under control of the same promoter (Prom.; ocher). After expression in E. coli, domains were co-purified with Ni-NTA coated agarose beads. (A) For western blotting, 0.08% of whole cell lysate (WCL) and flow-through fraction (FT), 0.2% of wash steps (W1, W5, W10), and 2% of eluates and bead fractions (E1, E2, BaE) were resolved on 10% SDS-PAGE. The bait CTD-HIS (above) was detected with antibody against HIS and the co-purified FLAG-NTD (below) with antibody against FLAG. (B) For Coomassie staining, 0.08% of whole cell lysate (WCL) and flow-through fraction (FT), 0.2% of wash steps (W1, W5, W10), and 3% of eluates and bead fractions (E1, E2, BaE) were resolved on 10% SDS-PAGE.

Since NTD and CTD of Pol5 expressed in trans restored wildtype growth of yeast cells (see Figure 51B), we cannot exclude that both domains interact with each other. To investigate this possible interaction, we cloned the sequences encoding both domains containing

their own stop codons in a single IPTG-inducible vector but separated by a shine-dalgarno sequence.

In addition, the NTD was fused to a FLAG tag at its N-terminus and the CTD fused to a HIS tag at its C-terminus (Figure 56). We affinity purified the Pol5-ΔN677 (or CTD) with Ni- NTA agarose beads and the fractions collected during the purification process were analyzed by western blotting (Figure 56A) and Coomassie staining (Figure 56B) (see 4.2.1.7 and 4.2.5). For purifications of single Pol5 domains from E. coli, we used a buffer with high sodium chloride concentration and without magnesium as indicated above. In order to do not disturb the association of both domains, we used buffer conditions based on the yeast affinity purification protocols (see also Table 9).

Western blot analysis with both antibodies (Figure 56A) revealed high amounts of co- expressed NTD and CTD (see WCL). The purification and elution of the CTD worked as efficiently (see E1, E2, and BaE with anti-HIS) as previously observed for the purification of the single domains (Figure 55). This result indicates that the new buffer conditions had no negative influence on the purification process. In addition, we could detect the NTD with anti-FLAG, in the elution fractions (see E2) and a few of it was retained in the bead fraction (see BaE). This result indicates a possible association of both domains, which might explain the rescue of cell growth in yeast depleted of Pol5 when expressed in trans. Since the antibodies against FLAG and HIS do not display the same sensitivity in western blot detection, no conclusion about a stoichiometric co-purification of the two domains is feasible. To have an idea about the stoichiometry of the interaction, the different fractions obtained from the purification process were also analyzed by Coomassie staining (Figure 56B). Unfortunately, we could not observe any band around 80 kDa, which may correspond to the NTD in the eluates or bead fraction (see E1, E2, and BaE). This result indicates the existence of a very small population of CTD associated with the NTD at least

in vitro. In addition, the result reflects a much higher sensitivity of the anti-FLAG antibody,