Our project divided protein expression and purification into two phases. The first hurdle that had to be overcome was the validation of the ToxAssay. Three proteins were needed. These were the LCwt, LCmut and SNAP25.

SNAP25

Synaptosomal-associated protein 25 (SNAP-25) is a t-SNARE protein. It is account for the specificity of membrane fusion in neuronal cells by forming the SNARE complex. It is almost exclusively formed in brain tissues and executes fusion by forming a tight complex that brings the synaptic vesicle and plasma membranes together.

The SNAP25 protein was provided with a Strep-Tag to obtain optimal results in purification. The other proteins (LCwt and LCmut) were each modified with a His-Tag. The reason for this is the Western-Blot Assay, which should show by means of Strep-Tag binding antibodies how the SNAP25 is degraded. However, this assay was rejected due to poorly binding antibodies.

The following ÄKTApurifier data were generated during purification:

The graph shows airborne noise at 25mL and SNAP25 peak at 77mL, since strep-tag bound proteins can be eluted very specifically.

LCwt

The wild-type light chain is a zinc endopeptidase that can cleave various proteins of the vesicle fusion apparatus (SNARE complex) and prevents exocytosis of the vesicles.

The LCwt was modified with a His tag. The hisTag may not be as specific as the Strep tag due to the presence of other histidine-rich proteins, but for this reason some washing steps were performed to elute non-specifically bound proteins as can be seen in the ÄKTApurifier data.

The following ÄKTApurifier data were generated during purification:

As can be seen in the graph, nonspecifically bound proteins were first removed by minimal addition of elution buffer (at 30mL and 42mL) and the LCwt was collected at 72mL in the fractions. This is a 4mL protein solution.

LCmut

The mutated light chain of botulinum neurotoxin C was mutated as follows: E446>A; H449>G; Y591>A. The resulting enzyme construct has no functional properties or enzyme activity and should not exhibit SNAP25 cleavage in the later ToxAssay.

The LCmut is tagged with a Polyhistidin-Tag for the same reasons as under point LCwt.

The following ÄKTApurifier data were generated during purification:

Nonspecifically bound proteins were first removed by minimal addition of elution buffer (at 50mL and 68mL) and the LCwt was collected at 95mL in the fractions.

In the second phase of protein purification, fusion proteins and their negative samples were purified to test different assays for the evaluation of our Shuttle system and to further characterize the proteins.

pHluorin2

pHluorin2 is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm. The protein is pH dependent.

The pHluorin2 is tagged with a Polyhistidin-Tag.

The following ÄKTApurifier data were generated during purification:

The graph shows an elution of the protein at even the smallest elution buffer concentrations. This should be taken into account when purifying pHluorin2 again to obtain more concentrated protein solutions. However, since the amount of protein was excellent, a second purification was not necessary.

The final protein concentrations and degrees of purity can be seen in the results - protein purification.

Results

To demonstrate the purity and expression of our various proteins, purification and expression assays were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration was determined via the nanodrop. The specific extinction coefficient of the respective protein was determined using the ExPASy ProtParam tool and the measurement was adapted accordingly.

SNAP25

Even though the expression was not very strong, SNAP25 was obtained even purer. The concentration was 2.01mg/mL which could be directly frozen to -80°C.

Goal of the chemical part of this years project was to alter the sodium channel blocker Eslicarbazepine so it would bind to the light chain of the detoxified Botox. The Synthesis was executeted after the following schematic plan.

The step-to-step execution of the synthesis can be found in our labbook. For the first step of our synthesis we achieved a satisfying yield after testing two diffrent reaction pathways. Massspectroscopy as well as NMR-Ananlysis showed that the exchange of the carboxy group with an thiol group has been sucessfull. The yield was determined to be approximatley 40%. The second step of the synthesis was executeted simultanousley with the synthesis of the azide-donor necessary for the last reaction. After some experiments to alter the reaction, we were able to successfully synthesize the azide-donor. The drying of the azide-donor was critical, as the donor decomposes in vacuo and under mild heat. As it’s a non-explosive and air-stable salt, maybe drying it in a desiccator could be a solution. Nonetheless, the Donor is instabel as long it’s not completely dry and must be handled with care. But by using the sulfate salt, the reaction became much more safe. The chloride salt should not be used, and the sulfate salt is an easy to make, shock-resistant alternative.

After executing the second reaction step the analytic results indicated that no reaction took place. Massspectroscopy data suggested that after the reaction both reactants were still present in their original Form as well as cleavage products of the 2,2'-Dithiodiethylamine. During troubleshooting we hypothesized that changing the conditions of the reaction would lead to a positive outcome. We considered Possible changes the reactiontime and -tempreature. The reactiontime might have been to short the activation energy of transitional state was to high. A possibel solution for the first problem would be to increase the reaction time. For the secon problem it would be to increase the reaction temperature. But with the already unstable 2,2'-Dithiodiethylamine, this might just destroy the reactants. A catalysator would be another option to decrease the activation energy. We also considered to take a whole diffrent approach. Because of the alkene as by-product, the yield was always very small. If the alkene was chosen as the product, targeting it with an S-Nucleophile, the yield could be improved. Plus, the disulfide binding could be produced simply by using a molecule with a disulfid binding. Due to a shortage of time, we were unable to test our possible solutions.

We tried to reobtain the reactants, but the Thio-Eslicarbazepine is not stable in water. The thiol group tends to decay, forming the alkene. As the solvent for the second reaction step was a water- THF-mixture, the Thio-Eslicarbazepine couldn't be reextracted in a sufficient amount. As a result we weren't able to test the Thio-Eslicarbazepine on our neuron-like cells to see if it's still preventing the sodium from entering, which would leave them less excitable. This would indicate that the changed Eslicarbazepine-SS-N₃ is still an active Na⁺-blocker.