Team:Freiburg/Results/Surface
Results: binding on surface
Optimizing cell-free expression as such is already a challenging task. But it becomes increasingly more complicated when the expressed proteins should be immobilized on a glass surface directly after expression: Cell-free mix is a complex system mainly consisting of proteins not that different to the target proteins to be immobilized. To overcome this hurdle we decided on fusing a tag to the proteins of interest that specifically binds on a catcher on the surface. But even with such a system, the surface has to be optimized in order to minimize unspecific binding.
Effective - The Unspecific Surface
As we needed a glass surface for measuring in iRIf, we used silanes to introduce reactive groups on the surface. Silanes integrate into the chemical structure of glass after being activated using oxygen plasma. Adding either the silane GOPTS (3-Glycidyloxypropyltrimethoxysilane) or APTES (3-Aminopropyltriethoxysilane) allowed us to couple certain chemical groups to the surface. The epoxy-group of GOPTS reacts covalently with amino-, hydroxy- and thiol-groups so that a large spectrum of molecules can be coupled. APTES on the other hand is not reactive itself but can be coupled to amino-groups with the homobifunctional cross-linker PDITC (p-Phenyldiisothiocyanate). For further information on chemistry and methods see surface chemistry methods . To evaluate these two coupling chemistries, we compared the binding capacities of the respective surfaces on simple microscope glass slides. Self-purified GFP in different concentrations was spotted on both surfaces and the resulting fluroescence intensity was compared (Figure 1). Especially for low concentrations it is clearly visible that APTES/PDITC outcompetes the GOPTS surface. Therefore, we continued our experiments using the APTES-chemistry. With these results we decided to switch on special glass slides, suitable for iRIf. With this method we were able to detect the interaction of anti-GFP antibodies with spotted GFP of different concentrations. To check for variations in different GFP-purification protocols, we compared GFP obtained from different groups (GFP1 to GFP3).
| GFP 1, 50 µg/ml | GFP 1, 17 µg/ml | GFP 1, 5 µg/ml |
| GFP 2, 50 µg/ml | GFP 2, 17 µg/ml | GFP 2, 5 µg/ml |
| GFP 3, 50 µg/ml | GFP 3, 17 µg/ml | GFP 3 ,5 µg/ml |
| bBSA (0.5 mg/mL) | BSA (10 mg/mL) |
Surface bound GFP was first washed mutliple times in the iRIf device. Anti-GFP in buffer solution was flushed over the slide and changes in surface layer thickness due to GFP-binding were detected in real-time. Figure 2 shows a quotient picture from this step, thus the increase in surface thickness due to antibody binding. The signal can further be amplified by a second binding step. As the anti-GFP antibody was biotinylated it could easily be attached to streptavidin flushed through the microfluidic chamber (Figure 3). This step also confirms the binding of anti-GFP to the spots and therefore the specificity of the interaction. Biotinylated BSA (bBSA, bottom middle) spotted on the slide, additionally served as positive control.
Getting Selective - The Specific Surface
All the surfaces presented above show, that a detection of antigen-antibody interaction is possible using a surface chemistry. But as we need a specific surface to purify antigens out of cell-free expression mix, we fused a 10x Histidin tag to our proteins and optimized a Ni-NTA (Nickel-Nitrolotriacetic acid) surface derivatization. To determine the specificity of this Ni-NTA surface we compared it to the PDITC surface. To asses the purifcation properties we used complete E. coli lysate transformed with either a tagged or an untagged GFP-construct. Additionally purified GFP-His as used in previous experiments was spotted and fluorescence intensity was measured. Figure 4 shows the intensities obtained for all spots: the intensity for the His-GFP lysate on the Ni-NTA surface is about four times higher than on the PDITC surface, while the values for the purified His-GFP are in a comparable range. The mean intensity for the untagged GFP-lysate spot is in the range of the background for the Ni-NTA surface and just slightly higher for the PDITC surface, which shows, that the GFP cannot bind to Ni-NTA without a His-tag. The low fluorescence intensities for lysate on PDITC may be due to the fact, that non-fluorescent proteins in the lysate are bound as efficient as GFP. This way the surface is blocked for further GFP molecules and overall fluorescence decreases compared to the specific Ni-NTA surface.
The next step to go, was testing the specific surface not only with E. coli lysate but with actual self-established cell-free expression mix. So we extended the experiment described above by spots with cell-free mix containing therein expressed GFP.
| # | Spot |
|---|---|
| 1-3 | Cell free expressed His-GFP |
| 4-6 | Cell free expression mix without DNA (neg) |
| 7-8 | His-GFP lysate |
| 9 | Untagged GFP lysate (unspecific binding) |
| 10 | bBSA (unspecific binding) |
| 11 | Purified GFP (1 mg/mL) |
The strong signal for the spots 1-3, as shown in figures 5 and 6, indicates the binding of anti-GFP antibodies to the cell free expressed GFP. So our specific surface enables us to enrich GFP on the surface to an amount detectable in iRIf. As already seen in previous experiments, the His-GFP lysate spots (7-8) showed a strong signal, while for the untagged GFP lysate (spot 9) and bBSA (spot 10) almost no signal was detected. In this experiment a common obstacle in iRIf measurement is visible: due to the formation of air bubbles in the microfluidic chamber, the high signal of purified GFP (spot 11) in only partially visible. Thus our surface is not only able to catch tagged proteins from a complex mixture, but also enriches these proteins on the glass slide, enabling detection in iRIf.
Diverse - Other Surface Systems
Besides Ni-NTA we worked with the Promega Halo-Tag system, to find the system best suited to our needs. The Halo-Tag that can be fused to target proteins binds covalently to chloralkanes which are immobilized on the surface. We tested several ligands, which differed in length of the alkane chain and surface attachment method. The ligand that worked best for us, was a 3-chloropropylsilane, which we directly immobilized on plasma activated iRIf slides. To test the surface we spotted our self-expressed Halo-GFP and Halo-mCherry as well as purified Halo-GFP as a positive control and purified untagged GFP as a negative control, which we both got from a research group from Osnabrück. As an additional negative control we spotted bBSA. The iRIf measurement showed that the Halo-tagged GFPs were successfully immobilized on the surface. Unfortunately there was a lot of unspecific binding, so that the negative control nearly bound as much to the surface as the positive control. The needed optimizations that would be necessary for a specific surface could not be performed due to time limitations. We decided to work with the Ni-NTA surface we established for future experiments.
Validation of our controls
Given that we were using purified GFP-His Tag for evaluation of our self-made surfaces and in further experiments as positive control we validated it by western blot. Therefore we showed the presence of GFP-His Tag with anti-His HRP Conjugate. Additionally we used the specific anti-GFP antibody as for the detection in iRIf.
