Best Measurement

Tau Phosphorylation & Aggregation Criteria

At first, we wanted to establish tau aggregations that is almost identical to that forming in vivo, believing that would be cost-effective, safer than using the animal model, and more importantly, would make it easier for future teams to use an optimized ready-to-use protocol with every part of approved from literature to be working efficiently.



Why have we used the phosphorylation procedure?


Thus, it is generally believed that tau proteins must undergo a sequence of biochemical and conformational changes before turning into misfolded substrates. Phosphorylation is the most common form of tau post-translational modifications found in vivo4. It has been suggested to be a pathological switch, leading to the formation of cytotoxic tau aggregates in NFTs5



Why did we use PKA and (SAPK4 OR GSK-3β) and didn’t use mainly heparin?


An early study has demonstrated that prior phosphorylation with cAMP-dependent protein kinase A (PKA) can prime the tau proteins for other kinases by inducing conformational changes to allow further phosphorylation events13. Moreover, a recent study that screens for the effect of 352 human kinases have shown that glycogen synthase kinase 3β (GSK-3β) and stress-activated protein kinase 4 (SAPK4) were the most active protein kinases phosphorylating tau at AD-specific epitopes that were recognized by phospho-tau specific antibodies including AT8 (pSer202 and pThr205), AT180 (pThr231), AT100 (pThr212 and pSer214), and PHF-1 (pSer396 and pSer404)10,14



Thus, we built a phosphorylation workflow that uses PKA followed by either GSK-3β or SAPK4 in a sequential manner to generate hyperphosphorylated tau with AD-specific epitopes in vitro.



We hypothesize that hyperphosphorylated tau could spontaneously assemble into small amorphous aggregates at physiological concentrations without using any external inducer. This is important since external inducers like heparin, the canonical method to generate in vitro tau aggregates, has recently been cast into doubt when examining recombinant and AD-derived aggregates of tau found differences in their conformations, properties, and activities15,16. We found that these hyperphosphorylated tau aggregates are more cytotoxic than the wild-type (WT) aggregates and can cause membrane destabilization and Toll-like receptor 4 (TLR4)-dependent responses in human macrophages.



Whereas, The heparin-induced WT tau first elongated into fibrillar aggregates with an apparent average length of 2400 ± 500 nm and then slowly fragmented into smaller aggregates, in agreement with the aggregation-and fragmentation pattern as previously observed26. On the other hand, g-tau and s-tau slowly polymerized into small aggregates with apparent average lengths of 530 ± 220 nm and 670 ± 400 nm, respectively. Fibrils above 1 μm were observed only rarely for s-tau. Critically, these hyperphosphorylated tau species remained as small aggregates over 300 h of incubation. Hence, we suggest that both hyperphosphorylated tau species could self-assemble to form pFTAA-active aggregates and that such aggregations of hyperphosphorylated tau were significantly slower than that of heparin-induced WT tau, consistent with previously reported study23.




Why did we use SAPK4, not GSK-3β?


We also found that PKA/SAPK4 had greater synergy than PKA/GSK-3β, not only in adding phosphate groups at more possible sites, as previously measured by our LC-MS/MS, but also in incorporating more phosphate moieties per tau molecule while all other reaction conditions are being controlled.



Procedure


Phosphorylation and aggregation of tau protein using kinases (PKA & SAPK4) protocol(hyperlinked experiment)
Phosphorylation of tau protein using (PKA & SAPK4) and aggregation using heparin protocol(hyperlinked experiment)
Tau aggregation using heparin only:We initiate the tau protein aggregation in 3 ways (phosphorylation only, phosphorylation and heparin, heparin only) and compare them using 5 assays.
1] ADP assay
2] SDS-PAGE
3] DLS
4] THT Assay
5] Transmission Electron Microscope (TEM)



ADP Assay Description


This assay measures ADP production from a kinase reaction by linked enzymes that first convert ADP to ATP and then quantify ATP using luciferase in the presence of luciferin. We used this assay to determine the efficiency of phosphorylation reaction.



SDS-PAGE Description


We use SDS-PAGE to test if the PKA & SAPK4 managed to add phosphate group to the tau protein. If the tau protein is phosphorylated successfully the band of phosphorylated tau will appear at size bigger than the tau protein only.



What will be loaded on the SDS-PAGE ?



1] Tau protein only (as a negative control)
2] PKA protein (to see the band of PKA will appear at which size)
3] SAPK4 protein (to see the band of SAPK4 will appear at which size)
4] Tau phosphorylated by PKA only (to test the phosphorylation efficiency of PKA)
5] Tau phosphorylated by SAPK4 only (to test the phosphorylation efficiency of SAPK4)
6] Tau phosphorylated by PKA and SAPK4 together (to test the phosphorylation efficiency of PKA and SAPK4)
Also, we used SDS-PAGE to characterize the aggregation test. If the tau protein successfully aggregated the band of aggregated tau will appear at size bigger than the tau protein only.

What should be loaded on the SDS-PAGE ?


1] The tau protein only (as a negative control)
2] PKA protein (to see the band of PKA will appear at which size)
3] SAPK4 protein (to see the band of SAPK4 will appear at which size)
4] The tau protein from aggregation by phosphorylation by PKA & SAPK4 and aggregated by incubation
5] The tau protein from aggregation by phosphorylation by PKA only and aggregation by incubation (to see the aggregation result from phosphorylation by PKA only)
6] The tau protein from aggregation by phosphorylation by SAPK4 only and aggregation by incubation (to see the aggregation result from phosphorylation by SAPK4 only)
7] The whole test (aggregation by PKA & SAPK4 and aggregation by incubation) without the tau protein (as a negative control)
8] The tau protein from aggregation by phosphorylation by PKA & SAPK4 and aggregated by heparin
9] The tau protein from aggregation by phosphorylation by PKA only and aggregated by heparin (to see the aggregation result from phosphorylation by PKA only and aggregation by heparin)
10] The tau protein from aggregation by phosphorylation by PKA only and aggregated by heparin (to see the aggregation result from phosphorylation by SAPK4 only and aggregation by heparin)
11] The whole test (aggregation by PKA & SAPK4 and aggregation by heparin) without the tau protein (as a negative control)
12] The tau protein from aggregation test by heparin
13] The heparin only (as a negative control) .



DLS Test Description



we used dynamic light scattering to determine the size of aggregated tau using 3 different ways:
1] Phosphorylation by PKA & SAPK4 and aggregation by incubation.
2] Phosphorylation by PKA & SAPK4 and aggregation by heparin.
3] Aggregation by heparin.
Tau protein.



THT Assay Description

Thioflavin-T is a dye that binds to β-sheet and its absorbance shift from 411nm to 280nm. So, if the aggregation reaction happened successfully the absorbance at 280nm will be very high.
We do this assay for:1] Aggregated tau from phosphorylation by (PKA & SAPK4) and aggregation by incubation.
2] Aggregated tau from phosphorylation by (PKA & SAPK4) and aggregation by heparin.
3] Aggregated tau by heparin only
4] Tau only (as a negative control)



TEM Test Description



we used transmission electron microscope to determine the size and the shape of tau aggregate after each test, so we tested:1] Phosphorylation by PKA & SAPK4 and aggregation by incubation.
2] Phosphorylation by PKA & SAPK4 and aggregation by heparin.
3] Aggregation by heparin.
4] Tau protein



Pulldown Assay

Description


pulldown assay is used to determine protein-protein interaction using GST or Ni-NTA column. If the protein binds successfully with the other protein, the SDS-PAGE gel will contain the bands of the 2 proteins will appear. It is an easy one-step protocol that could be used instead of ELISA and Western blotting



We do a pulldown assay to test the interaction between


- His-DOCs & GST-COH2 (at different time intervals to determine the best incubation time)
- His-COH2 & GST-DOCs
- His-Tau & GST-COH2-L-WWW (to test the binding affinity between tau protein and tau binding peptide WWW)
- His-Tau & GST-COH2-L-TD28rev (to test the binding affinity between tau protein and tau binding peptide TD28rev)
- His-TRIM21-L-DOCs & GST-COH2-L-WWW (to test the binding affinity between DOCs domain linked to TRIM21 and COH2 domain linked to Tau binding peptide WWW).
- His-TRIM21-L-DOCs & GST-COH2-L-TD28rev (to test the binding affinity between DOCs domain linked to TRIM21 and COH2 domain linked to Tau binding peptide TD28rev)
We not only used SDS-PAGE to determine the results of pulldown, but also we used the native-PAGE.

Ubiquitination Aassay

Description


pulldown assay is used to determine protein-protein interaction using GST or Ni-NTA column. If the protein binds successfully with the other protein, the SDS-PAGE gel will contain the bands of the 2 proteins will appear. It is an easy one-step protocol that could be used instead of ELISA and Western blotting.

This assay aims to add ubiquitin molecules to specific protein using E3 enzymes; we used this assay to test the activity and the specificity of our system to tau protein. If our system works perfectly, the tau-binding peptide will bind to tau. In the same time, the COH2 domain will bind tothe DOCs domain, as a ,result the TRIM21 will start in adding a ubiquitin molecule on tau protein and it will appear at bigger size when run on SDS-PAGE.

We will test the activity of TRIM21 by

1] Add all ubiquitination assay components with tau protein.
2] Add all ubiquitination assay components without E1 (as a negative control).
3] Add all ubiquitination assay components without E2 (as a negative control).
4] Add all ubiquitination assay components without ubiquitin (as a negative control).
5] Add all ubiquitination assay components without ATP (as a negative control).
6] Add all ubiquitination assay components without TRIM21 (as a negative control).

His-UBE2N His-TRIM21-L-DOCs His-UBE2w His-UBE2v2 GST-COH2-L-TD28REV GST-COH2 HIS-TRIM21 HIS-TAU GST-Coh2-WWW His-Docs GST-DOCs His-COH2

UBE2N


Description


Gene UBE2N (our part code). The ubiquitin-conjugating enzyme that this gene expresses belongs to the family of E2 enzymes. It serves as the second enzyme in the ubiquitination cascade to take the thioesterified ubiquitin from the E1 active site. Finally, by attaching to both the protein substrate and the E2-bound ubiquitin, the E3 ubiquitin ligase facilitates the transfer of ubiquitin onto the substrate. [3] To stimulate the synthesis of polyubiquitin chains, it interacts with UBE2V2 (E2 ligase). [2] According to in vivo research, this protein may also be involved in DNA post-replication repair. Hence,E2 ligase conjugates ubiquitin and participates in the ubiquitination process, leading to protein breakdown.


Ligation Reaction


we cloned our part (UBE2N) in pJET cloning vector (#K1231) and run ligated part on agarose gel (1%) and gel image indicated our Ligated protein and use this gel to know part size, vector size and part and vector after ligation.



pJET + UBE2N Transformation into DH5α

We transformed (UBE2N) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.



PGS + UBE2N Transformation into Bl21:


We transformed (UBE2N) Into BL21 bacterial cells to express this part as a protein and colonies which appears in the plate shows transformation done well and the efficiency of transformation was detected.



Affinity Chromatography

After protein induction happened, we had crude protein and we need to get our purified target protein so we go performed affinity chromatography to get the pure protein.


Result

BCA (after affinity) Comment: BCA #23225


After comparison induced and non-induced absorption results with standard curve our protein concentration is around (0.1 ϻg/ml).




References 1. Ncbi.nlm.nih.gov. 2022. UBE2N ubiquitin conjugating enzyme E2 N [Homo sapiens (human)] - Gene - NCBI. [online] Available at: [Accessed 13 October 2022]. 2. Shukla, P. K., Sinha, D., Leng, A. M., Bissell, J. E., Thatipamula, S., Ganguly, R., Radmall, K. S., Skalicky, J. J., Shrieve, D. C., & Chandrasekharan, M. B. (2022). Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 affect ubiquitination activity and decrease enzyme stability. The Journal of biological chemistry, 102524. Advance online publication. https://doi.org/10.1016/j.jbc.2022.102524 3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin–proteasome system. Trends in cell biology, 24(6), 352-359.

His-TRIM21-L-DOCs



Description


This fusion protein is part of our Trim System (Snitch System), it is a modified version of the NUDT 2020 part (BBa_K3396005). It is supposed to bind to the PROTAC (Coh2-linker-tau_binding_peptide) to assemble a full system that will be able to target Tau protein through the binding peptide and recruit ubiquitin to tag the whole protein to initiate degradation by proteasomes through Trim21. Trim21 (E3) is an integral part of the protein turnover processes which serves as a quality control step. In order to be degraded by proteasome 26S, the protein must be tagged with a ubiquitin tail. E3 ligase serves the function of transferring the ubiquitin to the protein of interest. So, in order for the process to be directed more specifically at certain proteins, we took advantage of the high affinity between the two modules (DocS and Coh2) which make up the cellulosome, to act as a protein pair that will guide trim to any chosen protein. This process could be done by anchoring Trim21 to either one of the modules and anchoring its other counterpart with a targeting domain for the protein of interest, more feasible and directed ubiquitination of the target protein has been achieved, which eventually leads to specific degradation.

Ligation Reaction

We cloned our part (His-TRIM21-L-DOCs) in pJET cloning vector (#K1231) and run ligated part on agarose gel (1%) and gel image indicated our Ligated protein and use this gel to know part size, vector size and part and vector after ligation.


Result


ligation between pJET and His-TRIM21-L-DOCs will appear at size 4203 on the gel.


pJET+His-TRIM21-L-DOCs Transformation into DH5α


We transformed His-TRIM21-L-DOCs Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.


His-TRIM21-L-DOCs protein extraction


We extracted His-TRIM21-L-DOCs using chemical lysis buffer composed from ((50 mM (Na2Hpo4/NaH2po4(Ph=8)/ (1mg/ml) Lysozyme/(25unit/ml) DNASE/(2mg/ml) protease inhibitor/ (300Mm) NaCl / (0.4%) Triton -X -100 / (10%) glycerol. Then we used BCA assay to quantify our protein.


SDS-PAGE of HIS-TRIM-linker-DOC


After extraction of Trim-linker-DOC protein, we performed SDS-PAGE to make sure the extraction went well, and to make sure that the induction time and concentration gives a significant yield


Affinity chromatography


After protein induction happened, we had crude protein, and we need to get our purified target protein, so we go performed affinity chromatography to get the pure protein.


Pulldown


We used pull-down assay to test the protein-protein interaction between TRIM21-L-DOCs & GST-COH2, and the GST-Coh2-L-WWW and GST-Coh2-L-TD28REV, then we made BCA assay to measure the quantity of the interacting protein then ran the elution in the gel.



References
1. Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698. 2. Ronchi, V. P., & Haas, A. L. (2012). Measuring rates of ubiquitin chain formation as a functional readout of ligase activity. In Ubiquitin Family Modifiers and the Proteasome (pp. 197-218). Humana Press. 3. Collins, G. A., & Goldberg, A. L. (2017). The logic of the 26S proteasome. Cell, 169(5), 792-806.

His-UBE2W




Description


The main member of the trio of enzymes, E2 ubiquitin-conjugating enzyme UBE2W, is in charge of TRIM21 E3 ligase's monoubiquitination, which serves as the catalyst for the enzyme's polyubiquitination by the heterodimer of UBE2N and UBE2V2. [1]. Ubiquitin is a highly conserved 76 amino acid polypeptide that requires the ATP-dependent activation of an E1 enzyme. Through a covalent link, the E1 binds the C-terminal end of ubiquitin to a cysteine residue in its active site. The second enzyme in the cascade to accept thioesterified ubiquitin from the E1 active site is E2, also known as the ubiquitin-conjugating enzyme. Finally, by binding to both the protein substrate and the E2-bound ubiquitin, the E3 ubiquitin ligase facilitates the transfer of ubiquitin onto the substrate [2].

pJET +UBE 2w Transformation into DH5α


We transformed (UBE2W) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and detect efficiency of transformation



pGS-21a+UBE 2w Transformation into BL-21


After protein induction happened, we had crude protein and we need to get our purified target protein so we go performed affinity chromatography to get the pure protein.


Affinity Chromotography


After protein induction happened, we had crude protein and we need to get our purified target protein so we go performed affinity chromatography to get the pure protein.


References

Stewart, M. D., Ritterhoff, T., Klevit, R. E., & brzovic, P. S. (2016). E2 enzymes: more than just middle men. Cell research, 26(4), 423-440. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin–proteasome system. Trends in cell biology, 24(6), 352-359.
Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin–proteasome system. Trends in cell biology, 24(6), 352-359.

His-UBE2v2

Description


This gene encodes a homolog of ubiquitin-conjugating enzyme E2 variant 1. These ubiquitin-conjugating enzymes don’t have the conserved cysteine residue critical for the catalytic activity of E2s. Based on the specific E2 used, the E2 enzymes can direct the ubiquitination process to different subsets of ubiquitin lysins. The formation of UBE2N/UBE2V2 complex facilitates the elongation of ubiquitination to forma a polyubiquitin chain. In our case, we will use its interaction with the UBE2N (E2 ligase) to catalyze the formation of polyubiquitin chains and the degradation of our targeted proteins.



pJET+UBE2v2 Transformation into DH5α


We transformed (UBE2V2) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.






pGS-21a+UBE2v2 Transformation into BL-21


We transformed (UBE2V2) Into BL21 bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.


Affinity Chromotography


After protein induction happened, we had crude protein and we need to get our purified target protein so we go performed affinity chromatography to get the pure protein



References 1.UBE2V2 ubiquitin conjugating enzyme E2 v2 [homo sapiens (human)] - gene - NCBI. (n.d.). Retrieved September, from https://www.ncbi.nlm.nih.gov/gene/7336 2.David, Y., Ziv, T., Admon, A., & Navon, A. (2010). The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. Journal of Biological Chemistry, 285(12), 8595-8604. 3.Pharos: Ubiquitin-conjugating enzyme E2 N (Tchem). (2022). 4.Vittal, V., Wenzel, D. M., Brzovic, P. S., & Klevit, R. E. (2013). Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer. Cell biochemistry and biophysics, 67(1), 103-110.

GST-COH2-LINKER-TD28REV


Description


This composite part serves massively in recognizing its counterpart which is Trim – DocS via the strong affinity and complementarity between the two proteins which are DocS and CoH2. Both systems combined can be used for directed ubiquitination for a specific protein of interest. Peptide TD28REV is used to direct the whole complex to the tau proteins, which is considered as one of the main causes of Alzheimer’s Disease. The system can be universally used against any protein of interest just by changing the binding peptide with another one that is designated to target the specific protein of interest.



Ligation of (PJET +(GST-COH2-LINKER-TD28REV))


To verify that we had a successful ligation of (GST-COH2-LINKER-TD28REV) into the blunt-ended plasmid (PJET), we check the size on gel electrophoresis (loaded 1µg of ligated plasmid).
Results show the size of the ligated plasmid is 4260 bp as expected but a faint band.





(PJET +(GST-COH2-LINKER-TD28REV)) Transformation into DH5α.



Number of colonies = 2400 colonies.

We transformed (PJET +(GST-COH2-LINKER-TD28REV) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.


Miniprep



(PGS +(GST-COH2-LINKER-TD28REV)) Transformation into BL21.


We transformed GST-COH2-LINKER-TD28REV Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and detect efficiency of transformation.




Number of colonies = 5 colonies



BCA assay before affinity.



After expression of protein into BL21, we extracted the protein. BCA assay is used to measure concentration of the protein.



Figure: Histogram shows differences in conc. between induced and non-induced samples.


Comment:
from the shown graph, the absorbance of total protein in the induced samples is higher than the non-induced. So, we expect that the high absorbance is due to our protein.


SDS-PAGE for comparison between induced and non-induced GST-COH2-L-TD28REV.




Pull-down assay between the tau protein and GST-COH2-L-TD28REV





References
Dammers, C., Yolcu, D., Kukuk, L., Willbold, D., Pickhardt, M., Mandelkow, E., Horn, A. H., Sticht, H., Malhis, M. N., Will, N., Schuster, J., & Funke, S. A. (2016). Selection and Characterization of Tau Binding ᴅ-Enantiomeric Peptides with Potential for Therapy of Alzheimer Disease. PloS one, 11(12), e0167432. https://doi.org/10.1371/journal.pone.0167432 journals.plos.org Selection and Characterization of Tau Binding ᴅ-Enantiomeric Peptides with Poten... A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein

GST-COH2

DESCRIPTION


CoH2 (Cohesin) is a cellulolytic enzyme in Clostridium thermocellum [1]. The Cohesin family has a great affinity for binding with its complementary counterpart family under the name of dockerins, with an essential role in the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome) [2] [4]. This interaction happens in two different forms, called the dual binding mode [3], in a calcium-dependent manner due to the presence of a calcium-binding site in the dockerin protein [4]. We used the DocS-Coh2 binding in our PROTAC system to conjugate E3 ligase trim 21 [BBa_K4165001] with the binding peptide to trigger the degradation of our targeted protein.



(GST-COH) + pJET cloning vector) Gel:




pJET+ GST-COH Transformation into DH5α



We transformed (GST-Coh2) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and the efficiency of transformation was detected.




Gel for miniprep product



After transformation, we extract our plasmid which contains our part GST-coh (plasmid miniprep) and then run miniprep product on the gel (1%) and the band appears with size (4209) which indicates miniprep done.



SDS PAGE for induced and non -induced (GST-COH)

After induction our protein with IPTG concentration (1mM) for (16hr). We extracted total protein with lysis buffer containing of:

*(50 mM (Na2Hpo4/NaH2po4(Ph=8))
*(1mg/ml) Lysozyme
*(25unit/ml) DNASE
*(2mg/ml) protease inhibitor
*(300Mm) NaCl
*(0.4%) Triton -X -100
*(10%) glycerol.

We ran the samples of the induced sample lysate and the noninduced sample to detect whether the chosen IPTG concentration and time intravel was right



BCA (lot: #SA242260) for total protein concentration



After comparison induced and non-induced absorption results with standard curve our protein concentration is around (0.1175 ± 0.02050)


Pull down assay for testing Gst-Coh2 and His Doc’s binding:


We perfomed pull down assay to test the binding between the two protein. To identify the amount of the interacting partners (GST-Coh2 and His-DOC), we made a BCA assay to measure the quantity of which.





References
1. brás, J. L., Carvalho, A. L., Viegas, A., Najmudin, S., Alves, V. D., Prates, J. A., Ferreira, L. M., Romão, M. J., Gilbert, H. J., & Fontes, C. M. (2012). Escherichia coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes. Methods in Enzymology, 510, 395-415. https://doi.org/10.1016/B978-0-12-415931-0.00021-5 2. Slutzki, M., Ruimy, V., Morag, E., Barak, Y., Haimovitz, R., Lamed, R., & Bayer, E. A. (2012). High-Throughput Screening of Cohesin Mutant Libraries on Cellulose Microarrays. Methods in Enzymology, 510, 453-463. https://doi.org/10.1016/B978-0-12-415931-0.00024-0 3. Stahl, S. W., Nash, M. A., Fried, D. B., Slutzki, M., Barak, Y., Bayer, E. A., & Gaub, H. E. (2012). Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proceedings of the National Academy of Sciences, 109(50), 20431-20436. 4. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, Bayer EA. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J Mol Recognit. 2009 Mar-Apr;22(2):91-8. doi: 10.1002/jmr.926. PMID: 18979459.

HIS-TRIM-21


DESCRIPTION


Tripartite motif-containing 21 (TRIM21) is an E3 ubiquitin ligase that has a strong affinity for the Fc domain of antibodies. It is mainly composed of four domains (RING domain, B-box domain, coiled-coil domain, and PRYSPRY antibody-binding region). TRIM21 engages the ubiquitin-proteasome system to destroy antibody-bound pathogens during infection. In our project, we used the truncated version of Trim21 proposed by team NUDT 2020 BBa_K3396007, they replaced the ‘PRYSPRY’ region with a protein pair (Protac), one of the pair will be fused to Trim21 and the other to our tau binding peptide, resulting in targeting and degradation of tau upon binding of the Protac.

(HIS-TRIM-21) + (PJET cloning vector) Gel




pJET+ (HIS-TRIM-21) Transformation into DH5α




We transformed (HIS-TRIM-21 )Into DH5α bacterial cells to amplify this part and colonies appear in the plates shows transformation done well and the efficiency of transformation was detected




Gel for miniprep product




After transformation, we extract our plasmid which contains our part (HIS-TRIM-21) (plasmid miniprep) and then run the miniprep product on the gel (1%) and the band appears with size (3943) which indicates miniprep is done.
miniprep concentration =




pGS-21a+ HIS-TRIM-21 Transformation into BL-21





We transformed (HIS-TRIM-21)Into BL21 bacterial cells to produce our protein in addition, the colonies that appear in the plate show transformation done well and detect the efficiency of the transformation




BCA for total protein concentration after protein extraction:



After induction our protein with IPTG concentration(1mM) for( 16 hr. )we extracted total protein with lysis buffer ((50 mM( Na2Hpo4/NaH2po4(Ph=8)/ (1mg/ml) Lysozyme/(25unit/ml)DNASE/(2mg/ml)protease inhibitor/ (300Mm) NaCl /(0.4%) Triton -X -100/(10%) glycerol.



HIS-TRIM-21 Concentration




BCA assay (lot: #SA242260) indicates that our protein of interest was eluted successfully in elution 1 sample with concentration around 0.084 mg/ml



References
1. Clift, D., McEwan, W. A., Labzin, L. I., Konieczny, V., Mogessie, B., James, L. C., & Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell, 171(7), 1692-1706.e18. https://doi.org/10.1016/j.cell.2017.10.033 2. D.L. Mallery, W.A. McEwan, S.R. Bidgood, G.J. Towers, C.M. Johnson, L.C. James Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21) Proc. Natl. Acad. Sci. USA, 107 (2010), pp. 19985-19990 3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends in cell biology, 24(6), 352. https://doi.org/10.1016/j.tcb.2013.12.003 4. L.C. James, A.H. Keeble, Z. Khan, D.A. Rhodes, J. Trowsdale Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 6200- 5. Zhang, Y., Li, L., Munir, M., & Qiu, H. (2018). RING-Domain E3 Ligase-Mediated Host–Virus Interactions: Orchestrating Immune Responses by the Host and Antagonizing Immune Defense by Viruses. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.01083

HIS-TAU



Description

Microtubule-associated protein (MAP), often known as tau, is a phosphoprotein frequently present in the cytosol and axons of neurons. It is found to be substantially expressed in ocular tissues and neurons of the central nervous system (CNS). It plays a critical function in the pathogenesis of Alzheimer's disease as well as in physiologically normal circumstances. It can maintain the neuronal microtubules in the healthy brain, which are necessary for intracellular signal transmission, the development of cell processes, and the establishment of cell polarity. A single gene on chromosome 17 that codes for six molecular tau isoforms that are produced by alternative splicing of tau pre-mRNA are known to be highly hydrophilic, heat stable, and soluble. The binding repeats of these six isoforms are either 3R taus or 4R taus, and the extra 4R repeat originates from the second (R2) repeat present in 4R. In 4R, there are two forms (0N4r and 2N4R), and both are intracellular and found in Alzheimer's disease, but in AD models, 0N4R is more preferable to use as its aggregates are easier and faster to form. Also, it is the closest model for AD disease, so we choose to work on it.



His-Tau size


we cloned our part (His-Tau) in pJET cloning vector (#K1231) and run ligated part on agarose gel (1%) and gel image indicated our Ligated protein and use this gel to know part size, vector size and part and vector after ligation.

Results
the band of His-Tau is expected to appear at size 1338 bp.





Comment

His-Tau part size is 1338 bp so the band is expected to appear between 1500 bp and 1000 bp as shown in the gel



part size and vector size(pJET+ His-Tau size)


Results
2974 for pJET + 1338 for His-Tau so expected size will be 4312 bp.



Comment

His-Tau part size is 1338 bp so the band expected to appear between 1500 bp and 1000 bp as shown in the gel.
And for pJET plasmid size is 2974 bp so the band expected to appear before 3000 bp band.



Ligation Reaction

By using T4 ligase we ligated the His-Tau part in pJET vector



Part size and vector size after ligation
Results
as a result of ligation reaction 2974 for pJET + 1338 for His-Tau so expected size will be 4312 bp.

Gel (Quantification of pJET+ His-Tau size)



Comment

His-Tau part size is 1338 bp so the band expected to appear between 1500 bp and 1000 bp as shown in the gel.
And for pJET plasmid size is 2974 bp so the band expected to appear before 3000 bp band.
So, for ligation of these two parts (plasmid + part) will be 4312 bp above 4000 bp band.



pJET + His-Tau Transformation into DH5α


We transformed (His-Tau) Into DH5α bacterial cells to amplify this part and colonies appearing in the plate show transformation done well and detected the transformation efficiency


The transformation of His-Tau into DH5α happened with high efficiency, so the plate is crowded because we plated 200μL.



Miniprep


After transformation we extract our plasmid which contain our part His-Tau (plasmid miniprep) and then run miniprep product on gel (1%) and band appears with size (4312) which indicate miniprep done.



Comment
After transformation we extract our plasmid pJET which contain our part His-Tau (plasmid miniprep) and then run miniprep product on gel (1%) and band appears with size (4312 bp (2974 for pJET + 1338 for His-Tau)) which indicate miniprep done.





pJET +His-Tau Transformation into Bl21


We transformed (His-Tau) Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and detect efficiency of transformation





His-Tau protein extraction


After induction of our protein with IPTG concentration (1mM) for (6hr), we extracted total protein with lysis buffer. The SDS PAGE gel image shows our proteins were successfully induced with the chosen IPTG concentration and time interval.



SDS-PAGE





BCA (before affinity)



Comment:
BCA #23225 which indicate total proteins with different size.



Affinity chromatography


After protein induction happened, we had crude protein and we need to get our purified target protein so we go performed affinity chromatography to get the pure protein.




BCA (after affinity)




comment
BCA #23225

After comparison induced and non-induced absorption results with standard curve our protein concentration is around (0.24866 ± 0.104462)



Tau Aggregation


We used heparin to aggregate the tau. as it was proved to form aggregates of differnet sizes in vitro.



Results



Comment

SDS-PAGE showed that induction of tau monomer found between 50 kDa and in case of forming aggregation as shown in this gel the tau aggregates which formed by heparin made different sizes of aggregates appear ranged from 100-200 kDa.



Pulldown


Using this assay to prove the interactions between proteins (His-Tau part interact with TD28 rev peptide.



Results



Comment

As shown interaction between (Tau aggregates) and two binding peptides (TD28 rev and WWW) have been successfully happened as it appear as a smear because we did not heat samples before loading.



BCA





Comment

AS this figure show pulldown assay prove interaction between (Tau aggregates) and two binding peptides (TD28 rev and WWW) have been successfully happened.



References
1- Iqbal, K., Liu, F., Gong, C. and Grundke-Iqbal, I., 2010. Tau in Alzheimer Disease and Related Tauopathies. Current Alzheimer Research, 7(8), pp.656-664. 2- Muralidar, S., Ambi, S., Sekaran, S., Thirumalai, D. and Palaniappan, B., 2020. Role of tau protein in Alzheimer's disease: The prime pathological player. International Journal of Biological Macromolecules, 163, pp.1599-1617. 3- R. Sajjad, R. Arif, A.A. Shah, I. Manzoor, G. Mustafa Pathogenesis of Alzheimer’s disease: role of amyloid-β and hyperphosphorylated tau protein Indian J. Pharm. Sci., 80 (2018), pp. 581-591, 10.4172/pharmaceutical-sciences.1000397

GST-coh2-L-WWW


Description


Due to the strong affinity and complementarity between the two proteins, DocS and CoH2, this composite part greatly aids in identifying its counterpart, Trim-DocS. Combining both techniques enables the targeted ubiquitination of a particular protein of interest. The tau proteins, which are thought to be one of the primary causes of Alzheimer's, are utilized to route the entire complex to the WWW. By replacing the binding peptide with a different one that is intended to target any defective protein aggregation, the technique can be utilized globally against any protein of interest.


Ligation Reaction


we cloned our part (GST-coh2-L-WWW) in pJET cloning vector (#K1231) and run ligated part on agarose gel (1%) and gel image indicated our Ligated protein and use this gel to know the part size, vector size, and part and vector after ligation. Expected result: 2974 for pJET + 1310 for Gst-coh2-L-WWW so the expected size will be 4284 bp.



pJET +Gst-coh2-L-WWW Transformation into DH5α

We transformed (pJET+ GST-coh2-L-WWW ) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and detect efficiency of transformation.



Miniprep

After transformation we extract our plasmid which contain our part pJET +GST-coh2-L-WWW (plasmid miniprep) and then run miniprep product on gel (1%) and band appears with size (4284 bp) which indicate miniprep done successfully.




(PGS + Gst-coh2-L-WWW) Transformation into Bl21

We transformed (GST-coh2-L-WWW) Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and the efficiency of transformation was detected.


Gst-coh2-L-WWW Protein Extraction

After induction our protein with IPTG concentration (1mM) for( 4hr )we extracted total protein with lysis buffer ((50 mM( Na2Hpo4/NaH2po4(Ph=8)/ (1mg/ml) Lysozyme/(25unit/ml)DNASE/(2mg/ml)protease inhibitor/ (300Mm) NaCl /(0.4%) Triton -X -100/(10% )glycerol and SDS PAGE gel image shows our proteins with different size.



Pulldown

Using this assay to prove the interactions between proteins (His-Tau part interact with TD28 rev peptide and WWW peptide.


Results

References
Seidler, P. M., Boyer, D. R., Rodriguez, J. A., Sawaya, M. R., Cascio, D., Murray, K., ... & Eisenberg, D. S. (2018). Structure-based inhibitors of tau aggregation. Nature chemistry, 10(2), 170-176.

His-Docs


Description


DocS (Dockerin) is a cellulolytic enzyme in Clostridium thermocellum. Each cellulosomal enzyme has one or more catalytic modules as well as a single dockerin module. The scaffolding is multifunctional, with the nine Cohesin modules integrating nine dockerin-bearing enzymes into the complex and the carbohydrate-binding enzymes. The complex is bound to the cellulosic substrate by the Cellulose Binding Domain (CBM), and the C-terminal dockerin module is implicated in this process. The interaction between Cohesin and Dockerin happens in two different forms, called the dual binding mode, in a calcium-dependent manner due to the presence of a calcium-binding site in the docking protein.


Ligation of (PJET + His-DOCs)

To verify that we had a successful ligation of (his-DOCs) into the blunt-ended plasmid (PJET), we checked the size on gel electrophoresis (loaded 1µg of ligated plasmid).

Gel (Qualification of Ligation of (pJET + His-DOCs)

(PJET + His-DOCs) Transformation into DH5α


We transformed (His-DOCs) Into DH5α bacterial cells to amplify this part and colonies appear in the plate shows transformation done well and efficiency of transformation was detected.



(pGS + His-DOCs) Transformation into BL21


We transformed (His-DOCs) Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and efficiency of transformation was detected.



BCA assay before affinity


After induction our protein with IPTG concentration (1mM) for (6hr) we extracted total protein with lysis buffer ((50 mM( Na2Hpo4/NaH2po4(Ph=8)/ (1mg/ml) Lysozyme/(25unit/ml) DNase/(2mg/ml)protease inhibitor cocktail/ (300Mm) NaCl /(0.4%) Triton -X -100/(10% )glycerol and SDS PAGE gel image shows our proteins with different size. Then, BCA assay is done to check the concentration of the extracted protein.




BCA assay after affinity chromatography


BCA assay is done to quantify concentration of His-DOCs protein after purification using.



Pull-Down


Pull-down is done to check the binding between His-DOCs and GST-COH2 (protein-protein interaction) then the concentration of purified linked proteins is detected by BCA assay.




References
1. Brás, J. L., Carvalho, A. L., Viegas, A., Najmudin, S., Alves, V. D., Prates, J. A., Ferreira, L. M., Romão, M. J., Gilbert, H. J., & Fontes, C. M. (2012). Escherichia coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes. Methods in Enzymology, 510, 395-415. https://doi.org/10.1016/B978-0-12-415931-0.00021-5
2. Slutzki, M., Ruimy, V., Morag, E., Barak, Y., Haimovitz, R., Lamed, R., & Bayer, E. A. (2012). High-Throughput Screening of Cohesin Mutant Libraries on Cellulose Microarrays. Methods in Enzymology, 510, 453-463. https://doi.org/10.1016/B978-0-12-415931-0.00024-0
3. Stahl, S. W., Nash, M. A., Fried, D. B., Slutzki, M., Barak, Y., Bayer, E. A., & Gaub, H. E. (2012). Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proceedings of the National Academy of Sciences, 109(50), 20431-20436.
4. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, Bayer EA. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J Mol Recognit. 2009 Mar-Apr;22(2):91-8. doi: 10.1002/jmr.926. PMID: 18979459.
5. Wojciechowski, M., Różycki, B., Huy, P.D.Q. et al. Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module. Sci Rep 8, 5051 (2018). https://doi.org/10.1038/s41598-018-23380-9

GST-DOCs



Description


Dockerin S protein tagged with Glutathione S-transferase, Dockerin S. module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Cohesin 2. The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions. This serves an essential role in the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome), this interaction happens in two different forms, called the dual binding mode, in a calcium-dependent manner due to the presence of a calcium-binding site in the dockerin protein.

We used the DocS-Coh2 binding in our Snitch system to f orm the PROTAC pair that will conjugate E3 ligase Trim 21 (BBa_K4165001) with the binding peptide for our targeted protein tau.

Result: the band of GST-DOCs is expected to appear at size 1031.



Ligation Reaction

Part size and vector size after ligation

we cloned our part (GST-DOCs) in pJET cloning vector (#K1231) and run ligated part on agarose gel (1%) and gel image indicated our Ligated protein and use this gel to know part size, vector size and part and vector after ligation.



Expected Result:
we expect that the T4 ligase will ligate our part (GST-DOCs) with the blunt end vector (pJET). Hence, it will appear at size 4005.

pJET + GST-DOCs Transformation into DH5α


We transformed (GST-DOCs) Into DH5α bacterial cells to amplify this part and colonies appearing in the plate show transformation was done well and the efficiency of transformation was detected.




Miniprep


After transformation we extract our plasmid which contain our part GST-DOCs (plasmid miniprep) and then run miniprep product on gel (1%) and band appears with size (4005) which indicate miniprep done.

Expected Result: we expect to extract the pJET + GST-DOCs and the resulted band will appear at 4005.


pGS-21a+GST-DOCs Transformation into BL-21


We transformed (GST-DOCs) Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and detect efficiency of transformation




Protein Extraction


After the previous flow we extracted the protein then tested its concentration using BCA Assay.




Pulldown


Pull down assay is used to test the binding affinity between GST-DOCs and HIS-COH2, then BCA assay is performed to test the concentration of the purified proteins to confirm the binding.





References
1. Brás, J. L., Carvalho, A. L., Viegas, A., Najmudin, S., Alves, V. D., Prates, J. A., Ferreira, L. M., Romão, M. J., Gilbert, H. J., & Fontes, C. M. (2012). Escherichia coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes. Methods in Enzymology, 510, 395-415. https://doi.org/10.1016/B978-0-12-415931-0.00021-5
2. Slutzki, M., Ruimy, V., Morag, E., Barak, Y., Haimovitz, R., Lamed, R., & Bayer, E. A. (2012). High-Throughput Screening of Cohesin Mutant Libraries on Cellulose Microarrays. Methods in Enzymology, 510, 453-463. https://doi.org/10.1016/B978-0-12-415931-0.00024-0
3. Stahl, S. W., Nash, M. A., Fried, D. B., Slutzki, M., Barak, Y., Bayer, E. A., & Gaub, H. E. (2012). Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proceedings of the National Academy of Sciences, 109(50), 20431-20436.
4. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, Bayer EA. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J Mol Recognit. 2009 Mar-Apr;22(2):91-8. doi: 10.1002/jmr.926. PMID: 18979459.
5. Wojciechowski, M., Różycki, B., Huy, P.D.Q. et al. Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module. Sci Rep 8, 5051 (2018). https://doi.org/10.1038/s41598-018-23380-9

His-coh2



Description


Cohesin type 2 is an enzyme that binds to its counterpart DocS (BBa_K3396000) to form a protein pair used for the assembly of our PROTAC system.

pJET and His-COH2 transformation in DH5α


We transformed (His-coh2) Into BH5-alpha bacterial cells to express this part as a protein which appears in the plate shows transformation done well and detect efficiency of transformation



pGS-21a and his-coh2


Result: the band of pGS-21a and his-coh2 is expected to appear at 5931 bp
We transformed (His-coh2) Into BL21 bacterial cells to express this part as a protein which appears in the plate shows transformation done well and detect efficiency of transformation

His-coh2 protein extraction

After extraction the protein, BCA is performed to test conc of the protein.



Affinity Chromatography

After protein induction happened, we had crude protein, and we need to get our purified target protein so we go through affinity chromatography technique.




Pulldown

After purifying our proteins, we have to check the binding between His-coh2 and GST-DOCs. So, we performed an interaction reaction overnight between them then pull-down assay is performed to purify both proteins attached to each other.




SDS PAGE

SDS page is used to make sure the binding between his-coh2 and gst-DOCs