Characterization of native eIF3 in the Wheat Germ Extract

Although all 13 eIF3 subunits could be expressed in E.coli and further purified, in vitro reconstitution was not successful. Possibly, eIF3 assembly requires additional factors or the plant eIF3 complex is comprised out of further subunits so far unknown. To address these questions, the native wheat eIF3 complex was analyzed in immunoprecipitation (IP) experiments and the wheat germ extract was fractionated in order to analyze either the whole eIF3 complex or sub complexes by Western Blot.

Both techniques require antibodies, which specifically recognize the wheat eIF3 subunits.

Thus, in corporation with Dr. Cathrin Enke, antibodies against all wheat eIF3 subunits were raised and purified. High yields of antibodies were obtained due to the immunogenic potential of wheat proteins.

2.1.3.1 Purification and characterization of antibodies against eIF3

Individually expressed and purified eIF3 subunits were used to immunize rabbits, thereby inducing the production of antibodies against the target proteins in the animal. The antibodies were purified from the rabbit sera using multiple columns containing different proteins to reduce the amount of cross-reacting antibodies in the final elution: (1)E.coli

2.1 Characterization of recombinant and native wheat eIF3 complex 29

lysate coupled column to remove antibodies recognizing potential purified and thus co-injectedE.coli proteins, (2) affinity tag coupled column to remove antibodies recognizing the tag of the fusion-protein injected into the rabbits (here H₁₄-MBP-TEV) tag, (3) antigen coupled column to accumulate antibodies recognizing the target protein. Instead of the entire target protein, also individual domains can be used as antigen. Bound antibodies were eluted by 0.1M glycine pH2.2, neutralized and precipitated with ammonium sulphate.

For analysis, precipitated antibodies were dissolved in 2xPBS buffer and a Western Blot was performed on wheat germ extract stripes using 1µg/ml of the purified antibodies (figure 2.3 A). As shown, antibodies against eIF3h, eIF3i, eIF3l and eIF3m are highly specific towards their target protein and do not show any cross-reactions to other proteins present in the wheat germ extract. Antibodies against eIF3e, eIF3f, eIF3g, eIF3j and eIF3k do show cross-reactivity, however the main signal correlates with the target protein.

Figure 2.3:Purification of antibodies against eIF3 subunits

(A) Antibodies against all 13 wheat eIF3 subunits were purified from rabbit sera as described in 4.2.5.2. (B) antibodies recognizing wheat eIF3c were further purified via individual eF3c domains to increase the sensitivity of the antibodies towards the target protein and to remove cross-reacting antibodies. Analysis was performed by Western Blot on membrane stripes containing wheat germ extract. After blocking the membrane, each stripe was incubated with 1µg/ml of the purified antibody. After over night incubation and thorough washing with 1xTBS, the secondary goatα-rabbit IRdye antibody was added (1:50.000) for 1h. After washing, the blot was analyzed using the Odyssey scanner (settings: 700nm (L2.0, 800nm (1.0))). FL: full length; ctrl: control membrane: the wheat germ extract containing stripe was incubated with the secondary antibody only.

Antibodies against the four largest eIF3 subunits eIF3a, eIF3b, eIF3c and eIF3d show cross-reactivity to a multitude of other proteins, either caused by degradation products of the target protein or by similar epitops in other wheat proteins. To reduce the amount

2.1 Characterization of recombinant and native wheat eIF3 complex 30

of cross-reactions, the antibodies were subsequently purified via individual domains of the target proteins. For analysis, wheat germ extract was blotted on nitrocellulose, cut into stripes and incubated with the domain specific antibodies. Figure 2.3 B exemplarily shows the purification of the eIF3c antibodies against individual domains of the wheat eIF3c proteins. Antibodies recognizing domains eIF3c459-491 and eIF3c815-936 are highly specific towards eIF3c and do not show any other cross reactivity whereas antibodies purified via the full length eIF3c proteins shows significant cross-reactivity with other proteins present in the extract.

Optimization of antibodies against eIF3a, eIF3b, eIF3d and eIF3j were performed as well.

This work was greatly supported by the lab rotation students Metin Aksu and Ayesha Khan.

2.1.3.2 eIF3 forms a stable complex in the wheat germ extract

To analyze the composition of the native wheat eIF3 complex, the in-house prepared wheat germ extract was rebuffered from a low salt HEPES based buffer system to 50mM Tris/HCl pH7.5, 500mM NaCl, 5mM MgAc, 1mM DTT and fractionated using a SD200 16/60 gel filtration column (Pharmacia).

The eluted fractions were loaded on SDS polyacrylamide gels and analyzed for their eIF3 subunit content by Western Blot using the previously described eIF3 specific antibodies (see figure 2.3 A). Figure 2.4 shows the gelfiltration profile and a schematic representation of the tested eIF3 subunit content in each fraction. eIF3b, eIF3c, eIF3m, eIF3h and eIF3k seem to form a stable complex in the wheat germ extract as they co-migrate through the column. Members of all described eIF3 subcomplexes (Zhou et al., 2008) were tested in Western Blot suggesting that also the additional, non-tested eIF3 subunits migrate simi-larly. eIF3j is an exceptional eIF3 subunit due to the fact that it only associates loosely to the complex (Fraser et al., 2004). Hence, it eluted later from the column. As control eIF2αas non-eIF3 subunit was tested as well showing that this subunit did not co-migrate with the tested eIF3 subunits but eluted later from the column.

To further validate that eIF3 forms a stable complex in the wheat germ extract and to identify possible additional subunits or assembly factors, immunoprecipitation exper-iments were performed. Recombinantly expressed and purified ProteinA was coupled to maleimide 4B Sepharose (produced in the lab by D. G¨orlich) via a reduced cystein at the C-terminus of the protein. Next, IgGs against eIF3 subunits were covalently attached and the beads were incubated with wheat germ extract rebuffered to 50mM Tris/HCl pH7.5, 500mM NaCl, 5mM MgAc, 1mM DTT and centrifuged to remove aggregates. After incu-bation, the flow-through was collected and the beads were washed. Bound proteins were eluted with 0.1M glycine pH2.2 and subsequently precipitated with trichloroacetic acid.

2.1 Characterization of recombinant and native wheat eIF3 complex 31

Figure 2.4:Native eIF3 forms a stable complex in the WGE

Wheat germ extract was rebuffered to 50mM Tris/HCl pH7.5, 500mM NaCl, 5mM MgAc, 1mM DTT and subse-quently pre-cleared by ultracentrifugation. The supernatant was loaded onto a SD200 16/60 gel filtration column with a constant flow of 1ml/min. For size estimation 0.5ml Gel filtration standard (BioRad) was loaded to the column under the same conditions. 1ml elution fractions were collected and analyzed for their eIF3 subunit content by Western Blot (antibodies used at 1µg/ml concentration). The distribution of eIF3 subunits in the individual fractions is shown schematically, eIF2αdistribution is shown in yellow.

Elution samples were loaded onto polyacrylamide gels, stained with Colloidal Coomassie, the resulting bands were cut and finally analyzed by mass spectrometry to identify the immunoprecipitated proteins. Initial experiments resulted in elution fractions containing many proteins binding nonspecifically to the resin. To improve the purity of the immuno-precipitation elution samples a pre-clearing step of the wheat germ extract via ProteinA beads without coupled IgGs was performed, which greatly improved the purity of the samples.

Figure 2.5 exemplarily shows the elution samples for immunoprecipitation experiments performed with antibodies against wheat eIF3b and eIF3c. The proteins eluted from the beads used for pre-clearing can be seen as well (pre-clearing), showing the vast amount

2.1 Characterization of recombinant and native wheat eIF3 complex 32

of proteins unspecifically binding to the resin. The bands in the IP elution fractions are labeled according to the mass spectrometry results, the direct target protein of the used antibody is highlighted in red. In both cases all eIF3 subunits except the loosely attached eIF3j subunit co-precipitated with the target protein, strongly arguing for a stable eIF3 complex in the wheat germ extract. In addition, elongation factor eEF1α was identified in both cases, the chaperone CPN60 was found as a major band in the eIF3b IP sample.

It remains to be elucidated if the chaperone is a complex constituent or co-precipitated due to cross-reactivity of the antibody.

Figure 2.5:Identification of the native wheat eIF3 complex

To identify the components of the native wheat eIF3 complex, immunoprecipitation experiments were performed as described in 4.2.5.4. To achieve a higher purity of the elution, the wheat germ extract was pre-cleared over ProteinA beads not containing any coupled antibodies (bound proteins shown in “pre-clearing”). The IP experiments were performed with ProteinA beads covalent attached to eitherα-eIF3b orα-eIF3c antibodies. Elution samples were loaded on 12% polyacrylamid gels and stained with colloidal Coomassie. The resulting bands were analyzed by Mass Spectrometry revealing all eIF3 subunits co-precipitating except eIF3j.

Both, the wheat germ extract fractionation and the IP experiments showed that the native eIF3 complex in the wheat germ extract is present in a stable form. All 13 proposed plant eIF3 subunits could be identified by mass spectrometry. The aim to find possible additional eIF3 subunits or factors being attached to the complex and thereby stabilizing it, was not successful. As already mentioned, the complex could not be reconstitution by mixing individual recombinant subunits, although these seem to be sufficient to form a stable