Team:Tuebingen/Demonstrate
Results
Success isn't about the end result, it's about what you learn along the way.- Vera Wang
Molecular Biology
Protein Expression
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
SNAP25 is a 25kDa large protein with an extinction coefficient (E) of 74000.
The expression and purification can be seen in the assays shown.
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.
LCwt
LCwt is a 51kDa large protein with an extinction coefficient (E) of 48250.
The expression and purification can be seen in the assays shown.
The induction of IPTG can be easily followed by the expression assay, but the purification is more difficult due to the instability of the light chains. These were denatured when dialysis was too long. Ultimately, a concentration of 0. 91 mg/mL could be achieved through efficient work and used in the Tox Assay.
LCmut
LCmut is a 51kDa large protein with an extinction coefficient (E) of 48440.
The expression and purification can be seen in the assays shown.
The purification and expression of LCmut was similar to that of LCwt. These also denatured during dialysis due to instability. However, more of the inactivated enzyme could be purified at a final concentration of 1.33 mg/mL.
Summary
After dialysis and shock freezing, all proteins used were analyzed again by SDS PAGE and their purity tested. The results are shown in the figure below:
In conclusion, it can be shown that all proteins could be successfully purified and were subsequently used for the ToxAssay, which showed that the native structure of the proteins was still retained.
Phase 2 proteins: pHluorin2
pHluorin2 is a 28kDa large protein with an extinction coefficient (E) of 22015.
The expression and purification can be seen in the assays shown.
As can be seen from the purification assay, pHluorin could be purified in large quantities. For dialysis 8mL 3. 2 mg/mL pHluorin2 was used which is more than sufficient for the following assays.
Cell Culture
Chemistry
The 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 executed 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 different reaction pathways. Mass spectroscopy, as well as NMR-Analysis, showed that the exchange of the carboxy group with a thiol group has been successful. The yield was determined to be approximately 40%. The second step of the synthesis was executed simultaneously 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 unstable as long it’s not completely dry and must be handled with care. But by using the sulfate salt, the reaction became much safer. 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. Mass spectroscopy 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 reaction time and -tempreature. The reaction time might have been too short the activation energy of transitional state was too high. A possible solution for the first problem would be to increase the reaction time. For the second 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 taking a whole different approach. Because of the alkene as a 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 disulfide 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.
Bioinformatics
Parts
pHluorin2
pHluorin 2 - BBa_K2689000pHluorin2 - BBa_K2689000
pHluorin2 is a ratiometric, pH-dependent GFP. It's excitation spectrum varies as the pH increases/decreases. This allows pHluorin2 to be used as an accurate biosensor. A special use case is the tracking of proteins that move between different cell compartments and encounter varying pH environments. Compared to pHluorin, pHluorin2 additionally shows higher fluorescence levels. It was developed by Matthew Mahon.
Syntaxin1A - BBa_K2689001
SNAP25 - BBa_K2689002
Omomyc - BBa_K2689003