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Results: binding on surface

Optimizing cell-free expression as such is already a challenging task. But it becomes increasingly complicated when the expressed proteins should be immobilized on a glass surface directly after expression: The Cell-free mix is a complex system mainly consisting of proteins not that different to the target proteins. To overcome this hurdle we decided to fuse a tag to the proteins of interest that specifically binds to a chemically treated surface. But to get such a tag-system to work, the surface has to be optimized in order to minimize unspecific binding.

Effective - The Unspecific Surface

Figure 1: Comparison of fluorescence intensity of bound GFP on GOPTS and PDITC surfaces

For measuring in iRIf the target proteins have to be fixed to a glass surface slide, therefore we used silanes to introduce reactive groups on the surface. These Silanes can bind to glass after reactive hydroxy groups were created through oxygen plasma activation. Adding either the silane GOPTS (3-Glycidyloxypropyltrimethoxysilane) or APTES (3-Aminopropyltriethoxysilane) allows 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 covalently fused 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 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 PDITC-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)

Immobilized GFP was first blocked in the iRIf device with 10 mg/mL BSA. Anti-GFP in buffer solution was flushed over the slide and leads to a change in the optical thickness at the GFP spots. The binding was detected in real-time. Figure 2 shows a quotient picture from this step, thus the increase in optical 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 that also binds to Streptavidine served as positive control.

Figure 2: iRIf quotient picture of spotted proteins after anti-GFP bindingstrong>

Figure 3: iRIf quotient picture of spotted proteins after additional Strep-Cy5 binding

Getting Selective - The Specific Surface

Figure 4: Comparison of specific Ni-NTA with unspecific PDITC surfaces>

All the results presented above show, that a detection of antigen-antibody interaction is possible with these surface chemistries. But as we need a specific surface to bind antigens out of cell-free expression mix, we fused a 10x Histidine 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 GFP lysate on PDITC is 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.

After we showed that we were able to bind his-tagged proteins specifically, we attempted to immobilize our cell-free expressed His-GFP-Lysate on our Ni-NTA surface. You can find the results for this crucial part of our project at the main results page.

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 the group of J. Piehler 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 controls 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.

Figure 7: Quotient picture for the Halo-surface measurement. Spot 1-4 show the binding of biotinylated anti-GFP to successfully immobilized self purified Halo-GFP. On spot 5 to 8 Halo-mCherry, but as no anti-mCherry was flushed over, no signal can be observed. On Spot 9 the positive control (Halo-GFP from J. Piehler) was pipetted and on spot 10 the negative control (His-GFP) both show a clear binding to anti-GFP. Also the second negative control bBSA (spot 11) shows a signal after Streptavidine was flushed over.

Figure 8: binding curve for the Halo-surface measurement. The binding curve shows the binding of biotinylated anti-GFP to all The GFPs on the slide (with and without Halo-Tag) and the binding of Streptavidine to the bBSA spot and all the GFP spots, because the anti-GFP that was flushed over before is biotinylated.

Validation of our controls

Figure 9: Western Blots of desalted elution of purified GFP protein with His-Tag. (A) To verify the prescence of purified GFP protein with a His -Tag we performed a Western Blot with anti-His HRP Conjugate (1:1000).(B) Western Blot of GFP-His with specific anti-GFP (1:2000) and anti-Goat HRP antibody (1:5000). The expected molecular weight is 28 kDa.

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.