Difference between revisions of "Part:BBa K4165009"

Latest revision as of 22:30, 13 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

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.

                              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 the 0N4R tau gene starting with cloning with the 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. Then, we started the expression of tau protein inside BL-21 using IPTG. We 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. To test 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.

Ligation of His Tau with pJET cloning vector

We used T4 ligase to ligate His Tau with pJET cloning vector so, we incubated His Tau with pJET overnight at 15°C.

         Figure 2. This figure shows the ligation between His Tau and pJET, 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.

Transformation of His Tau in DH-5 alpha using pJET cloning vector and in Bl-21a using pGS-21a expression vector

The transformation was performed using TSS buffer as it shows the best transformation efficiency compared to Calcium Chloride and a combination of Calcium Chloride with Magnesium Chloride. The transformation efficiency was calculated for both His Tau in the pJET cloning vector and in the pGS-21a expression vector and they were found to be 10000 no. of transformants/ug and 576000 No. of transformants/μg respectively.

                                   Figure 3. Transformed plate of His Tau + pJET.

Miniprep of pJET cloning vector containing His Tau

Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein so, we performed miniprep for pJET containing His Tau.

           Figure 4. This figure shows the miniprep of pJET cloning vector containing His Tau, The band appears with 
            size (4312 bp (2974 for pJET + 1338 for His-Tau)) which indicates that the miniprep was done

Transformation of His Tau in BL-21 using pGS-21a expression vector

                                    Figure 5. Transformed plate of His Tau + pGS-21a.

SDS PAGE of induced and non induced samples of His Tau

SDS-PAGE depends on the molecular weight of the protein. So, we used SDS-PAGE for His Tau to check if our protein is found in its exact size and to compare the induced and non-induced samples.

            Figure 6. This figure shows the comparison between the induced and non-induced samples of His Tau, 
            showing that our protein is induced effectively owing to our right choice of IPTG, time interval, and 
            concentration.

BCA assay results for His Tau

BCA assay is a technique that is performed to check the concentration of the protein and it depends on the color of the BCA working solution which is directly proportional to the concentration of protein

       Figure 7. BCA assay results of His Tau to check our protein concentration which is found to be 0.327679138.

Affinity chromatography of His Tau

Affinity chromatography is a technique used to purify the proteins, to get the purified protein without the cell lysate so, we performed Affinity chromatography for His Tau to get the Tau protein to characterize it before performing pull down assay.

       Figure 8. This figure shows the BCA assay results after the affinity chromatography, after comparison induced 
        and non-induced absorption results with the standard curve our protein concentration is around (0.24866 ± 
        0.104462)

Pull-down assay of Tau aggregates against GST Coh WWW and GST Coh TD28Rev

Pull-down assay is a one-step technique used to check the protein-protein interaction. Pull-down was performed to check the binding between the Tau aggregates with GST Coh WWW and GST Coh TD28Rev. Then we used the BCA assay to characterize the result of the pull-down. The results of pull-down show that the interaction between Tau aggregates with GST Coh WWW is better than that of Tau aggregates 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

           Figure 9. This graph shows the comparison of the pull-down assay between Tau aggregates with GST Coh WWW 
                                                     and GST Coh TD28Rev.

Statistical analysis of the GST Coh (L) WWW and the pulldown with the tau protein

     Figure 10. Statical analysis of the GST Coh (L) WWWW and pull-down of the GST Coh (L) WWW and the tau proteins show 
            that the pull down was successful in the Tau binding with significant efficiency to the WWW peptide.

References

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