Optimizing cell-free expression as such is already a challenging task. It becomes increasingly complicated if the expressed proteins ought to be immobilized on a glass surface directly after expression: The Cell-free mix is a complex system mainly consisting of proteins, amino acids and other molecules. All of them compete with the protein of interest for binding at the glass surface. To overcome this hurdle we decided to fuse a tag to the proteins of interest that specifically bind to a chemically treated surface. 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 mean fluorescence intensity of immobilized GFP on GOPTS and PDITC surfaces. The mean fluorescence for different concentrations of GFP on GOPTS is representet by the red bars. GFP on PDITC is represented by blue bars. The value for the mean intensity was calculated by averaging the intensities for 3 spots on 3 different slides respectively. The standard deviation is represented by error bars.

For measuring in iRIf the target proteins have to be fixed to a glass slide, therefore we used silanes to introduce reactive groups on the surface. These silanes can bind to siliciumdioxide (glass) after reactive hydroxy groups were created by oxygen plasma activation. Adding either the silane GOPTS (3-Glycidyloxypropyltrimethoxysilane) or APTES (3-Aminopropyltriethoxysilane) allows us to bind 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 regular microscope glass slides. Self-purified GFP in different concentrations was spotted on both surfaces and the resulting fluorescence intensity was compared (Figure 1). Especially for low concentrations it is obvious that APTES/PDITC has a higher binding capacity the GOPTS surface under the conditions tested. Therefore, we continued our experiments using the PDITC-based surface 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 hand-spotted GFP of different concentrations. To check for variations in different GFP-purification protocols, we compared GFPs obtained from different groups (GFP1 to GFP3). Figure 2 shows the pattern in which the different GFPs, the positive control biotinylated BSA (bBSA) and the negative control BSA were spotted, as well as the total amounts of protein, that were spotted.

The 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 3 shows a quotient picture from this step, thus the increase in thickness of the protein layer 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 4). This step also confirms the binding of anti-GFP to the spots and therefore the specificity of the interaction. The streptavidin binds also to the positive control bBSA.

Figure 2: Spotting pattern for iRIf measurement of different GFPs on PDITC.

Figure 3: Quotient picture of iRIf measurement for PDITC surface after anti-GFP step. Anti-GFP bound to all GFP spots on the slide. There is a slight difference between the different concentrations detectable. The positive control bBSA is not yet visible, because no streptavidin was flushed over to this point.

Figure 4: Quotient picture of iRIf measurement for PDITC surface after streptavidin step. streptavidin bound to the positive control bBSA as expected and also to all the GFP spots, because the anti-GFP is biotinylated.

Selective - The Specific Surface

Figure 5: Comparison of specific Ni-NTA with unspecific PDITC surfaces.The red bars show the mean fluorescence intensity for the different GFPs (His-GFP lysate, untagged GFP lysate, purified His-GFP) on PDITC. The blue bars represent the mean fluorescence intensity for the same GFPs on Ni-NTA. The grey bar represents the background for both surfaces.

All the results presented above show, that the detection of antigen-antibody interactions is possible with the PDITC surface chemistry. But as we need a specific surface to bind antigens out of our cell-free expression mix, we wanted to build up a Ni-NTA (Nickel-Nitrolotriacetic acid) surface and therefore genetically fused all our target proteins to a 10x histidine tag. We established a protocol for the Ni-NTA surface and optimized it. The protocol was optimized until the unspecific binding of other proteins from the cell free mix was reduced to a minimal. To determine the specificity of our Ni-NTA surface we compared it to the PDITC surface. To asses the purification properties we used complete E. coli lysate transformed with either a tagged or an untagged GFP-construct. Additionally, purified His-GFP that was used in previous experiments was spotted and fluorescence intensity was measured. Figure 5 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 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 demonstrated that we can successfully bind his-tagged proteins to our Ni-NTA surface, we attempted to immobilize our cell-free expressed His-GFP-Lysate on this surface. You can find the results for this crucial part of our project on the main results page.

Covalent - Other Surface Systems

On the way to find the surface best suited for our needs we also worked with the Promega HaloTag system. The HaloTag, that can be fused to target proteins, binds covalently to chloroalkanes which are immobilized on the surface. We tested several ligands, which differed in length of the alkane chain and surface attachment method. The ligand working best for us was a 3-chloropropylsilane, which we directly immobilized on plasma activated iRIf slides. To test the surface we pipetted 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. Both controls were obtained 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 at the negative control spots nearly as much binding to the surface as at the positive control spots. The optimizations that would be necessary to establish a specific surface based on the HaloTag system could not be performed due to time limitations. We decided to continue our work with the Ni-NTA surface which was established for future experiments.

Figure 6: 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 was spotted. 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 streptavidin was flushed over.

Figure 7: 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). Streptavidin binds to the bBSA spot but also to all the GFP spots, because the anti-GFP that was flushed over before is biotinylated.

Validation of Our Controls

Figure 8: Western Blots of desalted elution of purified GFP protein with His-tag. A: To verify the presence of purified GFP protein with a His-tag we performed a Western Blot with anti-His HRP Conjugate (1:1000). B: Western Blot of His-GFP 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 His-tagged GFP 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 His-GFP with anti-His HRP conjugate (Figure 8A). Additionally, we used the specific anti-GFP antibody (Figure 8B) as for the detection in iRIf.