Revision as of 02:16, 18 October 2018

Results

Success isn't about the end result, it's about what you learn along the way.- Vera Wang

Molecular Biology

The root of our project is the moleculare biology lab. We started to amplified:

our vector pet28a

inserts like wildtype light chain (wt LC); heavy chain (HC)

parts SNAP25; pHluorin2

The first barrier was the mutation of our wt light chain. Via a PCR we got three fragments. The Gibson assembly combined the vector pet28a with our three fragments. After a positive Transformation, we sequence our plasmid. After a lot of negative clones we reached a breakthrough.

We accomplished our mutated light chain to get ready for the protein expression. By means of the sequencing we proofed the mutation in the DNA sequence.

This agarose gel picture shows the successful cloning of the mutated light chain in vector pet28a. The seconde band shows the positive cloning of the Wt light chain in vector pet28a

Restriction digest of mut LC (A8) and Wt LC (F5)

Alignment of mut LC with wt LC - Highlighted sequence -> desired mutations

Now we were allowed to express our mutated light chain.

Restriction digest of pet28a-HC-mutLC-HiBit and pet28a-HC-mutLC-Omomyc

Finallly, we achieved a Plasmid with our vector pet28a and the inserts heavy chain, light chain and part. In this case we successfully assembled the HiBit part and the Omomyc part.

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.

Schematic reprasentation of the stepwise chemical alternation of the eslicarbamizine.

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 normaly used chloride salt should not be utilized, and the sulfate salt is an easy to make, shock-resistant alternative.

Draft of the sythesis of the azide-donor.

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 could be the reaction time and -temperature. The reaction time might have been too short or the activation energy of the transitional state was too high. A possible solution for the first problem could be to increase the reaction time. For the second problem, it would be possible 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.

Reactionstep two: the reaction was not successful

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

Our bioinformatics group successfully generated a structure of BoNTC using homology modeling. Moreover, we developed BERT, a powerful deimmunization workflow which we not only applied very successfully to the BoNTC binding domain, but also in an extensive collaboration with iGEM Paris-Bettencourt on a ovispirin/l-fucose Mutarotase fusion protein. To read more, go to our BioInfopage.

Parts

pHluorin2

BBa_K2689000

pHluorin2 is a ratiometric, pH-dependent GFP. Its 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.

While GFP itself shows a relatively stable fluorescence behavior across a wide pH range (at least too stable to be quantified easily), a look at the chromophore suggests that quite a lot of acid-base chemistry is influencing the fluorescence. Because those reactions are confined to the inner core of GFPs beta barrel structure, the solvent pH does not affect the chromophore in a easily quantifiable way. The idea behind pHluorins is to exploit these characteristics and change the structure of GFP in a way that it interfaces the internal reactions to the solvent pH so that it has a direct influence on their fluorescence behavior. For this purpose Miesenböck, et al. performed a directed mutagenesis approach in which they developed two forms of pH-dependent GFPs: Ecliptic and ratiometric pHluorin.

These pHluorins show a marked difference in their absorption spectra when encountering different ambient pH. The wild type form of GFP has a bimodal excitation spectrum with two peaks at about 395nm and 475nm. While the ecliptic variants of pHluorin show a decreased fluorescence signal with lower pH, the ratiometric pHluorins show a more complex pattern: With lower pH, the absorption at 395nm decreases while the absorption at 475nm increases. This is a key attribute for using it as a fluorescent pH probe.

Syntaxin1A

BBa_K2689001

Syntaxins are nervous-specific integral membrane proteins involved in membrane fusion. They possess a C-terminal transmembrane domain, a SNARE domain and an N-terminal regulatory domain. Syntaxin-1A is involved in ion channel regulation and synaptic exocytosis. It interacts with SNAP25 and Synaptobrevin-2 to form the SNARE complex. This complex formation brings vesicle membrane and cell membrane into close proximity, which leads to merging of both membranes. In neuronal cells, synaptic vesicles store neurotransmitters that are released upon stimulation of the cell. This exocytosis process is, among other things, is regulated by Syntaxin1a. Syntaxin-1A is a known substrate of botulinum neurotoxin C, a protein with metalloprotease activity which prevents the exocytosis of neurotransmitters into the neuromuscular junction. Consequently, this leads to flaccid paralysis symptoms of the illness botulism.

SNAP25

BBa_K2689002

Synaptosomal-associated Protein 25 (SNAP-25) is a t-SNARE protein involved in the regulation of membrane and vesical fusion. It is integrated into the membrane via palmityl side chains and faces the cytosolic side. Together with Syntaxin-1A and Synaptobrevin-2, it forms the SNARE complex, in which it contributes 2 a-helices. This complex brings together vesicle membrane and cell membrane, which leads to the fusion of both. In neuronal cells, this leads to the exocytosis of neurotransmitters into the interneuronal junction. SNAP-25 is a known substrate of botulinum neurotoxin A, C and E, a protein with metalloprotease activity which prevents the exocytosis of neurotransmitters into the neuromuscular junction. Consequently, this leads to flaccid paralysis symptoms of the illness botulism.

Omomyc

BBa_K2689003

Omomyc is a dominant-negative mutant of Myc, a family of proto-oncogenes encoding for transcription factors which regulate growth, proliferation, tumorgenesis, and apoptosis. Myc dimerizes with Max over its basic helix-loop-helix zipper domain. This heterodimer then binds to the DNA enhancer box (E-Box) sequence and thus regulates the expression of corresponding genes. In cancer Myc often is constitutively expressed, leading to increased proliferation signals and thus to excessive tumor growth. Omomyc possesses a mutated basic helix-loop-helix zipper domain, in which 4 amino acids were exchanged: S57T, E64I, R70Q and R71N. These mutations lead to an altered dimerization behavior of the protein. Omomyc efficiently forms homodimers and furthermore also forms heterodimers with wild-type Myc, leading to a decreased formation of Myc/Max dimers. Accordingly, Omomyc works as a suppressor of Myc/Max binding to E-box elements and leads to a suppressed activation of artificial E-box promoter elements. In the past it was shown that Omomyc can selectively trigger apoptosis in cells overexpressing Myc, however the exact mechanism remains unclear. Researchers proposed that this happens through the transcriptional repression of specific genes. Through its ability to induce apoptosis in cells overexpressing Myc, as various cancers do, Omomyc may be subject to new therapeutic opportunities in the future.

Assays

HBit Nano Glow

To evaluate whether small peptides can be delivered into a neuronal cell via BoNTC, we expressed HiBit in frame with the light chain. This peptide is part of a split luciferase and has a very high affinity to the 18kDa LargeBit (LgBit) (for further information on the HiBit nanoluciferase visit the protein constructs page). The functional luciferase produces a strong luminescence signal at very low concentrations (> 1pM) that can be detected in a luminescence assay. Since we faced multiple complications in the purification process, we first tested all purification fractions on HiBit concentration by adding the luciferase substrate and LgBit to 50µl protein fraction. The resulting luminescence was quantified in a TecanM200 Infinite Pro plate reader showing that HiBit still binds to LgBit when fused to BoNTC and is not impaired in its luciferase function. Further the highest concentration of BoNTC-HiBit was found in the wash fraction 1 (Figure 1) which is why we decided to use this fraction for the following evaluation of BoNTC’s shuttle efficiency in our cell culture model.

Figure 1: HiBit Nano-Glo luminescence assay in five different protein fraction of the purification process. 50µl of each purification fraction was incubated with 0,5µl LgBit, 50µl reaction buffer and 1µl Nanoluciferase substrate for 15 minutes at 4°C. Values are presented as relative luminescence units (RLU).

The cell based luciferase assay was designed in a way that BoNTC-HiBit is first incubated with the cells for a 60 minutes duration to allow endocytosis. The cells are afterwards rinsed two times with PBS to remove residual extracellular BoNTC-HiBit. Thus, only intracellular HiBit is able to lead to a luminescence signal. Following the washing steps, the cell membranes are broken up to release HiBit into the extracellular environment. Finally, LgBit and the luciferase substrate are added to generate a luminescence signal detectable by the plate reader. While BoNTC-HiBit should be able to enter the neuronal SHSY5Y cells, mouse embryonic fibroblast cells (MEF) are also tested for a concentration of BoNTC-HiBit, since their cell membranes partly consist of gangliosides but should not allow a endocytosis of BoNTC-HiBit.

Figure 2: HiBit Nano-Glo luminescence assay in lysats of undifferentiated SHSY5Y and mouse embryonic fibroblast (MEF) cells. 30.000 cells of the respective cell line were incubated with different concentrations of BoNTC-HiBit (displayed on x-axis) for one hour at 37°C. Cells were then lysed by addition of 0,1% Triton-X100, and mixed with 200µl reaction buffer, 200µl PBS, 2µl LgBit and 4µl Nanoluciferase substrate. The samples were thereafter incubated for 15 minutes at 37°C. Values are presented as relative luminescence units (RLU), error bars are displayed as SEM.

The overall low luminescence compared with the values presented in Figure 1 can be explained by an a lower concentration of BoNTC-HiBit in relation to the well’s area, since the fractional analysis was executed in a 96 well plate while the cellular lysis assay was conducted in a 24 well plate. The luminescence values from the conducted luminescence assay increase when the cells are incubated with higher concentrations of BoNTC-HiBit. This confirms that BoNTC-HighBit retains its features as a split nanoluciferase with LgBit after 60 minutes incubation time. The interpretation of the assay itself allows multiple conclusions: The first explanation for an increased luminescence is the aforementioned successful endocytosis. Simultaneously, BoNTC-HiBit might get stuck on the surface of the cellular membrane and remain there bound to a membrane compartment. It also might be possible that the fusion protein bound unspecifically to the well material and could not be sufficiently removed in the washing process. On this basis, there is yet to be refinement done on this assay to find a reliable proof for a successful HiBit delivery into the cell.

pHluorin Assay

pHluorin2 is a GFP with the pH-dependent fluorescence characteristic of pHluorin and the superior efficiency of EGFP making it an ideal tool for studying endocytotic pathways in cells.

Important Data:

Absorption maximum 1: 395 nm

Absorption maximum 2: 475 nm

Emission maximum: 509 nm

When carrying out protein tracking experiments with pHluorin one has no ability to differentiate the cause for a decreasing fluorescence signal. While a possible reason can be a decrease pH, several other effects influence the signal strength. Those can be changing protein concentrations (e.g. caused by degradation), or fluorescence quenching due to long exposure times. To actually identify a decrease in pH, a single signal value is not enough. This is where ratiometric proteins come into play. Rather than using just one signal measurement to estimate the ambient pH, the observer performs two measurements at a different wavelength. Because the preferred excitation wavelength of pHluorin changes with pH, the ratio of the signals is an indicator of pH unaffected by the confounding effects described above.

To test the absorption maxima based on pH, we expressed the protein coding sequence in Lemo21 E.coli cells and purified the cells via a HisTrap FF column.

For the fluorescence measurement, we used the purified protein in PBS with a buffer capacity of 20 µM. We analogously prepared a PBS buffer with a buffer capacity of 70 µM and titrated it to pH values from 5.3 to 7.5. 80 µl aliquots of the second buffer were given into a White Opaque PerkinElmer CulturPlate-96. For a given pH three samples were prepared as triplets in three separate wells. After that, we added 20 µl of our protein solution (with a concentration of about 0.5mg/ml).

We measured fluorescence using a Tecan M200 Infinite Pro plate reader with the following settings:

Excitation Scan Excitation Wavelength Start 340 nm

Excitation Wavelength End 520 nm

Excitation Wavelength Step Size 3 nm

Emission Wavelength 550 nm

Integration Time 2 0µs

Number of flashes 25

Gain 50

This measurement yielded an emission spectrum (averaged over each three wells) for a pH range of 2,2 in 0,3 increments.

Figure 1: representative traces of an emission spectrum of pHluorin2 in a range of pH 5,3 to 7,5, values are presented as absolute fluorescence count with SEM

Based on the traces from the emission spectrum we saw emission maxima at 415 nm and 475 nm, differing quite drastically from previously reported data (J.Mahon, 2011). While fluorescence values increased with rising pH at 415 nM, fluorescence values decreased at more basic pH at 475 nm.

Figure 2: Emission maxima at 415 nm and 478 nm , values are presented as absolute fluorescence count with SEM

From our measurements, we calculated the ratio of associated 415 nm/475 nm maxima values leading to an approximately linear correlation in the chosen pH range.

Figure 3: 415 nm/ 478 nm ratio of total fluorescence counts in dependance to pH, values are presented as quotients of total fluorescence counts

Tox Assay

ToxAssay To provide a safe work environment for everyone involved in the process of protein purification and BoNTC based assays, we had first to prove, that our mutated botulinum toxin would not be able to cause neurotoxic cleavage of Snap25. Since the BoNTC light chain (LC) alone can not enter a cell, we expressed and purified the LC to test its cleavage ability compared to the wildtype LC. Based on the papers on findings of Vadakkanchery et. al and Edwin J.Vazquez-Cintron et al. we developed an assay which would show the activity difference between the purified LC wildtype compared to our LC mutant. The principle of the assay was the catalytic cleavage of the SNAP25 protein. SNAP25 cleavage by an active LC, which the assay was based on, results in a peptide fragment with the following amino acid sequence:

N’ATKMLGSGWSHPQFEK’C (Mpeptide: 1803 [g/mol]).

We chose a buffer for our cleavage assay composed of 150mM K+ glutamate and 10mM HEPES, adjusted pH to 7.3 with NaOH.

The incubation took place for over 18 hours at 37°C. Analytic samples were drawn from the reaction at the beginning of the assay labeled P0 and after the 18 hours labeled P1.

In order to obtain an optimal and unambiguous result, we selected the most sensitive detection methods available, Matrix-assisted laser desorption/ionization (MALDI). According to our knowledge, we were the first to show a successful SNAP25 cleavage assay via MALDI-TOF measurement.

MALDI, combined with mass spectrometry, is a special procedure that enables the analysis of peptides, proteins and oligonucleotides through gentle ion generation. The analytical sample is embedded in a suitable matrix, which shows a high absorption at the irradiated laser wavelength, whereby not only higher intensities of the analyte molecule ions are obtained, but almost no fragment ions occur. Since the sensitivity of such mass spectrometric techniques extends into the femtomol and subfemtomol range, this method was selected to provide us with clear results regarding our ToxAssay.

This very high accuracy is also reflected in following graphs:

As displayed, each time the MALDI shows a peak at 1804 m/z generated through SNAP25 cleavage by LCwt which fits the molecular weight of described peptide. The consistency of peak occurance in an indicator for LC enzymatic activity and maintenance of its protein function. In contrast, none of the samples incubated with LCmut show the described peptide fragment at 1804 m/z, which leads us to the conclusion that this enzyme was successfully deactivated via the triple mutation and thus leads in combination with the heavy chain to an inactivated, nontoxic botulinum neurotoxin.

To make sure, we detected the correct cleavage product, we further validated our results using a peptide synthesized specifically for this purpose as a positive control with the aforementioned peptide sequence. The peak displayed when only measuring the synthetic peptide, proved to be exactly the peak seen in the samples incubated with LCwt and therefore fortifies our conclusion of a successful detoxification.

References

Mahon, M. J. (2011) pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Advances in Bioscience and Biotechnology. (Print), 2(3), 132–137.. http://doi.org/10.4236/abb.2011.23021
Vaidyanathan, V. V., Yoshino, K. I., Jahnz, M., Dörries, C., Bade, S., Nauenburg, S., ... & Binz, T. (1999) Proteolysis of SNAP‐25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme‐substrate complexes and cleavage. Journal of neurochemistry, 72(1), 327-337..
Mahon, M. J. (2011) pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Advances in Bioscience and Biotechnology. (Print), 2(3), 132–137.. http://doi.org/10.4236/abb.2011.23021
Vazquez-Cintron, E. J., Beske, P. H., Tenezaca, L., Tran, B. Q., Oyler, J. M., Glotfelty, E. J., ... & Band, P. A. (2017) Engineering botulinum neurotoxin C1 as a molecular vehicle for intra-neuronal drug delivery. Scientific Reports, 7, 42923..
F. Lottspeich u. H. Zorbas. (1998) Bioanalytik. (Spektrum Akademischer Verlag F. Lottspeich u. H. Zorbas (Hrsg.).. http://doi.org/10.4236/abb.2011.23021

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+{{Tuebingen/ReferenceEntry|id=pH|authors=Vaidyanathan, V. V., Yoshino, K. I., Jahnz, M., Dörries, C., Bade, S., Nauenburg, S., ... & Binz, T. |title=Proteolysis of SNAP‐25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme‐substrate complexes and cleavage.|year=1999|journal=
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Difference between revisions of "Team:Tuebingen/Demonstrate"

Revision as of 02:16, 18 October 2018

Results

pHluorin2

Syntaxin1A

SNAP25

Omomyc

HBit Nano Glow

pHluorin Assay

Tox Assay

References