Samenvatting
Summary BMW Moleculaire Technieken eindverslag
Het eindverslag over het practicum van moleculaire technieken.
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Voorbeeld van de inhoud
The function of the TPR domain on the phosphataseactivity of the enzyme PP5 in Rattus norvegicus
December 2022
Charlotte van der Voort
Abstract
The protein PP5 is a phosphatase and regulates cell processes like proliferation and apoptosis. The
protein PP5 contains a TPR domain, that causes phosphatase activity if the domain binds to another
TPR domain. Hsp90 is a protein with a TPR domain, that causes dissociation of the TPR domain after
binding. The function of the TPR domain on the phosphatase activity is unknown. For that reason, the
function of the TPR domain on the phosphatase activity for PP5 in Rattus norvegicus is investigated.
This has been examined by comparing the phosphatase activity of the PP5 with full length and the
phosphatase activity of the PP5 without a TPR domain. The phosphatase activity was according to the
results by the PP5 without a TPR domain lower than the PP5 with full length. Because of this result it
can be concluded without certainty that the TPR domain stimulates the phosphatase activity of PP5 in
Rattus norvegicus inhibits.
Introduction
Phosphorylation and dephosphorylation of proteins are important posttranslational modifications that
regulate biological pathways and functions, like intracellular signal transduction (Yang et al., 2005).
Problems in the signal transduction can lead to cancer. PP5 also regulates biological functions in a cell
like proliferation, differentiation and apoptosis (Hinds and Sanchez, 2008). Phosphoprotein
phosphatase (PPP) belongs to the biggest class of the protein phosphatases. The PPP contains a highly
conserved catalytic subunit that will dephosphorylate serine and threonine residues. (Farkas et al.,
2007). Protein phosphatase 5 (PP5) belongs to the PPP family and contains a regulatory
tetratricopeptide repeat (TPR) domain on the N-terminal and a phosphate domain on the C-terminal
(Chinkers, 2001). The PP5 and the PPT (yeast homolog) are the only protein phosphatases with a TPR
domain (Hinds and Sanchez, 2008). One of the well-known functions of PP5 is that it activates the
checkpoint ATM kinase in DNA damaged cells (Ali et al., 2004, as cited by Yang, et al., 2005). If the
checkpoint ATM kinase is inactive it could cause cancer, because the cell cycle stops (G0 phase) and
apoptosis takes no longer place. Other PPP family members have a high standard activity in
comparison to PP5. That is likely because the reduced standard activity in PP5 is a result of the
regulatory TPR domain (Yang et al., 2005).
The TPR domain of PP5 has been shown to interact with the chaperone protein heat shock protein 90
(Hsp90) and consequently stimulates the phosphatase activity of PP5 (Ramsey and Chinkers, 2000, as
cited by Yang et al., 2005). In previous research was shown that the catalytic subunits and the TPR
domain of PP5 contains a binding groove of Hsp90 in the TPR domain. For this reason it is suggested
that the binding of Hsp90 causes dissociation of the TPR domain on the N-terminus from the
phosphatase domain on the C-terminus and thus the activation of PP5 (Yang et al., 2005). This would
suggest that the TPR domain inhibits the phosphatase activity of PP5.
, Therefore, the function of the TPR domain in PP5 will be investigated on the phosphatase activity in
Rattus norvegicus (RnPP5). Because of the results from the previous researches it is expected that the
TPR domain stimulates the phosphate activity of PP5 from Rattus norvegicus inhibits.
It is also expected that the phosphatase activity of the PP5 protein full length (PP5-FL) is lower than
the phosphatase activity of the PP5 protein deletion mutant without the TPR domain (PP5-dTPR).
Consequently, the research will be conducted by amplifying the PP5 protein full length (PP5-FL) and
the PP5 protein deletion mutant without the TPR domain (PP5-dTPR) using recombinant DNA
technology. The phosphatase activity of PP5 in E.coli with or without the TPR domain will be
investigated using a phosphatase assay.
Figure 1: The domains of the PP5 protein. This figure is made with snapgene.
Material and method
PCR amplification of PP5 FL and PP5-ΔTPR cDNA
To amplify the PP5-FL and PP5-ΔTPR fragments the following primers were used: pGEX-F (5’-
GCAGGGCTGGCAAGCCACGTTTGGTG-3’); dTPR-Rn-F (5’-CGGGATCCGTCGTGG
ACTCTCTGGACATCG-3’) and pET-32a-R (sequence 5’-
GCTCAGCGGTGGCAGCAGCCAACTCAGC-3’). The PCR was performed on 10 ng
pGEX/pET21a-RnPP5 using a PCR-mix containing 0.4 µM forward primers, 0.4 µM reverse primers,
10 mM dNTPs, 0.004 U/µl Phusion polymerase diluted in 5x HF buffer and made up to 300 µl with
MQ water. The annealing temperature of the primers was 61°C. A standard PCR program with 30
cycles was used. A negative control without DNA template was included. The amplified PCR products
were analyzed using gel electrophoresis with a 1% agarose gel.
DNA purification of PP5 FL and PP5-ΔTPR cDNA
To purify the PP5-FL and PP5-ΔTPR fragments a column was used. The DNA solution is first
extracted with a PB buffer (binding buffer) so the DNA binds to the column material, whereas the
impurities do not. The DNA was pelleted by centrifugating at 13000 rpm for 60 seconds. The most
impurities are removed. Briefly, following washing with 700 µl PE buffer (wash buffer) to remove the
remaining impurities using a QiaQuick column. The DNA was pelleted by centrifugation at 13000 for
60 seconds and resuspended in sterile water so the DNA is released from the column. A negative
control without DNA template and a positive control with undigested pET28a was included. The
purified DNA was measured with the Nanodrop.
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