All eIF3 subunits can be expressed and purified in a recombinant

tem-perature, the duration of expression, the E.coli expression strain, the induction strength, but also the N-terminal fusion tags. To determine the optimal set of parameters for each subunit, the proteins were cloned into eight different vector backbones, yielding fusion proteins with the following N-terminal tags: H₁₄, H₁₄-TEV, H₁₄-brSUMO, ZZ-TEV, H₁₀ -ZZ-TEV, H₁₄-shGFP-brSUMO, H₁₄-MBP-TEV and H₁₄-MBP-brSUMO. H₁₄- and ZZ-tag are well established affinity ZZ-tags, binding either complexed Ni²⁺ ions or IgGs. TEV (Tobacco Etch Virus) and brSUMO are protease cleavage sites, which are routinely used for tag - free protein purification (Frey and G¨orlich, 2014a). shGFP (super hydrophilic GFP) and MBP (maltose binding protein) are rather large tags with a molecular weight of ∼40kDa and a stable fold. MBP itself is known to show chaperone-like qualities when fused to the N-terminus of a protein (Pryor and Leiting, 1997; Smyth et al., 2003).

Table 2.1:eIF3 subunit solubility greatly depends on N-terminal fusion tag

All 13 eIF3 subunits were fused to different N-terminal tags. The constructs were expressed at 18℃over night in E.coliTop10F’ cells orE.coliNEB Express cells, induced with 100µM IPTG. The cultures were harvested, lysed and ultracentrifuged. The supernatant after ultracentrifugation was analyzed by SDS-PAGE for soluble protein. The solubility of the expressed constructs was rated from insoluble (red) over low solubility (orange), moderate solubility (yellow) to high solubility (green), meaning almost 100% of the expressed protein was produced in a soluble form.

H14- H14-TEV H14 eIF3a insoluble insoluble insoluble insoluble insoluble insoluble low low

eIF3b moderate high high moderate moderate moderate high moderate

eIF3c high high high high high high high high

eIF3d moderate high high high moderate high high high

eIF3e insoluble insoluble insoluble moderate low moderate moderate high

eIF3f insoluble insoluble insoluble moderate low high high high

eIF3g insoluble insoluble moderate moderate insoluble high moderate high

eIF3h moderate moderate low moderate low moderate high high

eIF3i insoluble insoluble insoluble moderate insoluble insoluble moderate low

eIF3j moderate high high high high high high high

eIF3k high high high high moderate high moderate high

eIF3l insoluble insoluble insoluble insoluble insoluble insoluble low low

eIF3m insoluble insoluble insoluble insoluble low moderate high high

The constructs were transformed into E.coli Top10F’ cells and expressed over night at 18℃. Expression was induced by the addition of 100µM IPTG, which releases repression by lacI, thereby enabling transcription of the downstream genes. The cells were subsequently harvested, resuspended in buffer (50mM Tris/HCl pH7.5, 500mM NaCl, 5mM MgAc, 1mM DTT), lysed by sonication and ultracentrifuged. Soluble protein is expected to remain in the supernatant after centrifugation, whereas protein aggregates precipitate to the pellet.

2.1 Characterization of recombinant and native wheat eIF3 complex 25

The supernatant samples were analyzed by SDS-PAGE to estimate the solubility of each construct. The results are summarized in table 2.1.

The solubility of eIF3 subunits was also screened at higher temperatures and shorter expression times (23℃, 6h). Although some eIF3 subunits such as eIF3c, eIF3d and eIF3j could be expressed in a soluble manner, the majority of the subunits tends to express better when induced at lower temperatures for a longer time period (data not shown).

Figure 2.1:Solubility of eIF3 subunits greatly depend on N-terminal fusion tags

eIF3 subunits eIF3a, eIF3c and eIF3m were expressed as fusion constructs with various N-terminal affinity tags.

Expression was performed inE.coliTOP10F’ cells at 18℃over night with 100µM IPTG (for details see 4.2.2.3).

Samples were taken after lysis of the cells by sonication (aL) and from the supernatant after ultracentrifugation (aUZ). Soluble protein should remain in the supernatant whereas insoluble proteins should have gone to the pellet.

As control, non-inducedE.colicells were loaded as well (bi). (*) indicates the expressed protein bands. All samples were loaded at a concentration of 30mOD/lane on a polyacrylamide gradient gel and stained with Coomassie.

shGFP: super hydrophilic GFP variant

In general, one can observe an order of solubility (1) between the individual eIF3 subunits and (2) between the different affinity tags used. When analyzing the solubility of the eIF3 subunits under all tested conditions, subunits eIF3b, eIF3c, eIF3d, eIF3j, and eIF3k are soluble with any tag (see table 2.1 and figure 2.1 eIF3c), indicating a stable fold of the individual proteins. eIF3h behaves similar to those subunits, however in comparison, expression and solubility levels are slightly lower. Subunits eIF3e, eIF3f, eIF3g, eIF3i, and eIF3m can not be expressed in a soluble manner with small tags, but the soluble fraction increases with increasing tag size (see 2.1 eIF3m). Subunits eIF3a and eIF3l show severe solubility problems. Both can only be expressed as partly soluble proteins when fused to MBP (see 2.1 eIF3a).

When grading the affinity tags for their ability to increase solubility of their fusion partners,

2.1 Characterization of recombinant and native wheat eIF3 complex 26

the rule of thumb seems to be: the larger the fusion tag, the more soluble protein can be produced (see figure 2.1 eIF3m and table 2.1). However, there are some exceptions. When comparing ZZ-TEV and H₁₀-ZZ-TEV, the slightly larger H₁₀-ZZ-TEV tag is not able to positively influence the solubility of e.g. eIF3g or eIF3i, whereas ZZ-TEV-fusion proteins show a moderate solubility. Possibly the biochemical properties of the histidine sidechains interfere with the folding of these particular eIF3 subunits. Another interesting phenomena is that although the H14-shGFP-brSUMO, H14-MBP-TEV and H14-MBP-brSUMO tags are all similar in size, only fusions to MBP enable to produce eIF3a and eIF3l in a slightly soluble manner. This might be explained by the chaperone-like qualities of MBP.

These screenings show that affinity tags can enhance the solubility of their fusion partners and that there seems to be a correlation between tag size and soluble fraction of the expressed protein. However, it also shows that the ideal tag for each protein has to be individually determined to perfectly match and positively influence the biochemical properties and folding kinetics of the protein.

Figure 2.2:Purification of eIF3 subunits by protease elution

eIF3 subunits eIF3c and eIF3k were N-terminally fused to an H14-brSUMO-MBP tag and expressed inE.coliNEB Express cells at 18℃over night with 200µM IPTG (for details see 4.2.2.3). Samples were taken before induction (bI), after induction (aI), after lysis by sonication (aL) and from the supernatant after ultracentrifugation (aUZ).

The lysate was applied to 6% Ni²⁺-chelate 500 ˚Asilica and after binding in batch for 1h, the flow through (FT) was collected. The resin was thoroughly washed and incubated with 0.1µM SENP1 protease cutting at the brSUMO protease recognition site. After incubation for 1h, the cut protein was washed out. As control, the remaining protein on the matrix was eluted with 1M imidazole. The full length construct is marked with green asterisks, the cleaved protein with red asterisks and the remaining tag is marked with light blue asterisks. All samples were loaded at a concentration of 30mOD/lane on a polyacrylamide gradient gel and stained with Coomassie.

2.1 Characterization of recombinant and native wheat eIF3 complex 27

In summary, expression conditions yielding in soluble proteins could be determined for each of the 13 eIF3 subunits, allowing to further purify the subunits by affinity chromatography as shown in figure 2.2 exemplarily for subunits eIF3c (left panel) an eIF3k (right panel).

The proteins were expressed as H₁₄-MBP-brSUMO constructs and elution was performed by on-column protease cleavage using brSENP1 protease (purification scheme can be seen in figure 1.5). Due to cleavage, the eIF3 subunit looses its affinity to the matrix and elutes, whereas the remaining tag (here H14-MBP-brSUMO) remains bound to the beads and can only be released from the Ni²⁺-beads with high imidazole concentrations, which displaces the His-tag and thereby the bound protein.