Difference between revisions of "Part:BBa K4165009"
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than that of Tau aggregates with GST COH TD28Rev | than that of Tau aggregates with GST COH TD28Rev | ||
===Pull down assay between td28rev and tau=== | ===Pull down assay between td28rev and tau=== | ||
| − | + | SDS was performed after the pull down assay to check the protein-protein interactions | |
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/tau-agg-tau-td28.jpeg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/tau-agg-tau-td28.jpeg" style="margin-left:200px;" alt="" width="500" /></p> | ||
</html | </html | ||
| + | Figure 6. This figure shows that the binding between TD28REV and Tau happened as there are two bands in the gel | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Revision as of 15:41, 12 October 2022
Tau (0N4R)
This basic part encodes the human microtubule-associated tau protein isoform 0N4R.
Usage and Biology
Alzheimer's disease (AD), which is considered the most common neurodegenerative disease to cause dementia, is characterized by 2 main accumulations that are amyloid plaques from amyloid beta and NFTs aggregates resulting from hyperphosphorylated or abnormally phosphorylated tau protein accumulation. Our part Tau, microtubule-associated protein (MAP) is a phosphoprotein that is prevalently found in cytosol and neuron axons. It is determined to be significantly expressed in neurons of the central nervous system (CNS) and the ocular tissues. It has a crucial role both under normal physiological conditions and also in the pathology of Alzheimer's disease.
In the normal brain, it can stabilize the neuronal microtubules that are essential for the establishment of cell polarity, the development of cell processes, and intracellular signal transduction. Six molecular tau isoforms are coded by a single gene on chromosome 17 resulting from alternative splicing of tau pre-mRNA and characterized to be significantly hydrophilic, heat stable, and soluble. These six isoforms differ in their binding repeats either 3R taus or 4R taus microtubule-binding repeats and the extra 4R repeat comes from the second (R2) repeat found in 4R. Tau biological activity is affected by 2 main processes that are alternative splicing and phosphorylation. For tau's interaction with tubulin and the enhancement of microtubule assembly, normal brain tau appears to require 2-3 moles of phosphate per mole of the protein. However, tau is phosphorylated at Ser262 and Ser214 in AD, which causes tau to separate from microtubules.
In the AD brain, tau Hyperphosphorylation is considered the main cause of AD progression, it may alter the protein's shape and charge, which in turn causes the microtubule-binding domain to become exposed and allow tau to self-assemble and form oligomers characterized to be neurofibrillary tangle. According to several studies, the polymerized tau (neurofibrillary tangles) is inert since it does not bind to tubulin or encourage its assembly into microtubules.
Before the development of NFTs, all six forms of tau are self-assembled into paired helical filaments as a result of hyperphosphorylation at the C-terminus of tau (PHFs). The aggregated tau protein takes this shape, which impairs axonal transit and continuously promotes microtubule instability. AD patients have an aberrant or hyperphosphorylated tau protein concentration that is four times higher than that of normal controls. These misfolded tau proteins are also recognized as potential neurotoxins and lose their basic function of MT stability along with enhanced aggregation effects.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 43
Illegal AgeI site found at 229 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 207
Illegal BsaI site found at 1119
Illegal SapI.rc site found at 393
Dry Lab Characterization
Modeling
The structure of Tau was modeled by several tools and the top model was retrieved from trRosetta ranking 5 out of 6 according to our quality assessment code.
| cbeta_deviations | molprobity | ramachandran_favored | ramachandran_outliers | Qmean_4 | Qmean_6 |
|---|---|---|---|---|---|
| 0 | 3 | 95.28 | 1.05 | -1.52844 | -2.316739 |
Figure 1. Predicted 3D structure of Tau(0N4R) modeled by trRosetta
WetLab Results
The tau protein is a protein responsible for the stabilization of microtubules that are essential for the establishment of cell polarity, the development of cell processes, and intracellular signal transduction. In AD tau is hyper-phosphorylated and that results in a change in his confirmation and starts to aggregate. We used 0N4R tau protein starting with cloning with pJET vector inside DH5 alpha bacterial cells. then we extract the plasmid to restrict the tau gene and ligate it with pGS-21a to transform it into BL-21. We started the expression of tau protein inside BL-21 using IPTG, then extract the tau using two different methods, physical and chemical methods. After lysis, we purify tau using Ni-NTA affinity chromatography. Then we begin to build a tau aggregate model by incubation with heparin. We tested the binding affinity between tau protein and two tau binding peptides which are TD28rev and WWW using pull-down assay, and to characterize the interaction we used the BCA assay.
Transformation of His Tau in DH-5 alpha using pJET cloning vector
We transform the tau protein into DH5 alpha using a pJET vector for cloning. We used TSS buffer for transformation as it shows the best transformation efficiency compared to Calcium Chloride and a combination of Calcium Chloride with Magnesium Chloride. The transformation efficiency = 10000 no. of transformants/ug and CFU/ml = 20000
Figure 2. Transformed plate of His Tau + pJET.
Transformation of His Tau in BL-21 using pGS-21a expression vector
Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 576000 No. of transformants/μg and CFU/ml = 1152000
Figure 3. Transformed plate of His Tau + pGS-21a.
SDS PAGE of induced and non induced samples of His Tau
We tested the induction efficiency using IPTG by SDS-PAGE and compared the bands between the induced and non-induced samples.
Figure 4. This figure shows the comparison between the induced and non-induced samples of His Tau,
where well no.2 is the non-induced sample while well no.5 is the induced sample showing that our
protein is induced effectively owing to our right choice of IPTG, time interval and concentration
Pull down assay of Tau aggregates against GST COH WWW and GST COH TD28Rev
We checked if our tau binding peptides are able to bind to tau aggregate using BCA assay and SDS-PAGE.
Figure 5. This graph shows the comparison of pull down assay between Tau aggregates with GST COH WWW and GST COH
TD28Rev, showing that the interaction between Tau aggregates with GST COH WWW is better than that of Tau
aggrergates with GST COH TD28Rev as the concentration of elution of Tau aggregates with GST COH WWW is more
than that of Tau aggregates with GST COH TD28Rev
Pull down assay between td28rev and tau
SDS was performed after the pull down assay to check the protein-protein interactions
