Three E. coli BL21 bacteria cultures containing GST, GST-ICP0, and 6xHis-USP7 proteins in Erlenmeyer flasks were prepared prior to the beginning of lab day 1. On lab day 1, the three bacteria cultures were collected and 1mL of each were transferred into plastic cuvettes. The cuvettes were placed in a spectrophotometer and the light absorption for each sample was recorded at 600nm. The bacteria cultures were placed in a shaking incubator at 37⁰C for one hour. When the incubation was completed, GST-ICP0 was transferred into a microfuge tube and was placed in a centrifuge for two minutes at 13000 rpm at room temperature. GST-ICP0 was labeled as Sample 1. A BSA standard curve was produced in Figure 1 using the Bradford Assay to determine the concentration of the purified protein samples. On lab day 2, Ni+2 Affinity Chromatography was preformed to purify the 6xHis-USP7 protein. Lysis/Binding Buffer, containing 5mL of Common Buffer (50mM Tris-HCl PH 7.5, 500mM NaCl) and 5µL of 5M Imidazole, was prepared. Wash Buffer, containing 9mL of Common Buffer and 27µL of 5M Imidazole, was prepared. Elution Buffer, containing 5.7mL of Common Buffer and 300µL of 5M Imidazole, was prepared.
A bacterial pellet containing USP7 was collected and 1mL of Ni+2 Lysis/Binding Buffer was added. The bacterial cells were vortexed and lysed at 30% intensity with four, 20 second pulses, and 15 second gaps. A Ni+2 IMAC column was prepared and 25µL of resin bed was added. 50µL of Ni+2 IMAC agarose resin (50% slurry) was added down the column. The column was equilibrated with Ni+2 Lysis/Binding Buffer and was rinsed three times. The 6xHis-USP7 was centrifuged for 10 minutes at 13000 rpm at 4⁰C. The 6xHis-USP7 bonded with the resin as 1 mL of the bacterial lysate was added to the column. The column was sealed and incubated for 30 minutes at 4⁰C. 10µL of the 6xHis-USP7 lysate was collected in a separate microfuge tube and was labeled as Sample 2. After incubation, the resin was centrifuged for 1 minute at 1000 rpm at room temperature. 8mL of Ni+2 Wash Buffer was added to the resin column in 1mL increments. To detach 6xHis-USP7 from the resin, 200µL of Ni+2 Elution Buffer was added to the column. The concentration of the eluted 6xHis-USP7 protein was recorded using the Bradford Assay. The optical density was measured using a spectrophotometer at 595nm. 400µL of the 6xHis-USP7 elution was placed in 1x PBS Buffer with a PH 7.4 for 48 hours at 4⁰C to be dialyzed. Glutathione Affinity Chromatography was performed to purify the GST-ICP0 protein. 1mL of 1x PBS with PH 7.4 was added to the GST-ICP0 bacterial pellet. The pellet was vortexed and then transferred to a microfuge tube to be lysed in a sonicator at 30% intensity with four, 20 second pulses, and 15 second gaps.
The GST-ICP0 lysate was then placed in a microcentrifuge for 10 minutes at 13000 rpm at 4⁰C. 100µL of resin bed and 200µL of Glutathione-agarose resin (50% slurry) was added to a column in preparation of the glutathione affinity column. The column was equilibrated as 1mL of 1x PBS with PH 7.4 was added to rinse the column three times. To bind the GST-ICP0 lysate with the resin, 250µL of the lysate and 750µL of 1x PBS with PH 7.4 were added to the column and the column was incubated for 30 minutes at 4⁰C. 10µL of the GST-ICP0 lysate was collected and labeled as Sample 3. After incubation, the resin was centrifuged for one minute at 1000 rpm at room temperature and 7mL of 1x PBS with PH 7.4 was added in 1mL increments. The resin was labeled GST R and GSTI R and were Sample 4 and 5 respectively. On day 3, 20µL of the dialyzed 6xHis-USP7 was collected and labeled as Sample 6. GST and GST-ICP0 protein columns were collected and washed twice with 1mL of 1x PBS with PH 7.4. 200µL of the purified and dialyzed 6xHis-USP7 elution and 800µL of 1x PBS with PH 7.4 was transferred to the GST and GST-ICP0 columns. The columns were sealed and were incubated for 90 minutes at 4⁰C. After incubation, 20µL of the two columns were collected and labeled GST PD-UP (pull-down unbound proteins) and GSTI PD-UP. GST PD-UP was Sample 7 and GSTI PD-UP was Sample 8. GST and GST-ICP0 columns were washed with 6mL of 1x PBS with PH 7.4.
The columns were labeled GST-PDW (pull-down wash) and GSTI-PDW. 20µL of each were collected and labeled Sample 9 and Sample 10 respectively. GST and GST-ICP0 columns were eluted with 200µL of GST Elution Buffer (20nM reduced glutathione in 50mM Tris-HCl PH 9.0). The two columns were labeled GST PD-Elt (pull-down elution) and GSTI PD-Elt. 20µL of each were collected and labeled as Sample 11 and Sample 12 respectively. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel was casted from a 4% stacking gel and a 15% SDS polyacrylamide separating gel. On lab day 5, 750mL of 1x SDS-PAGE Running Buffer was prepared using 75mL of 10x stock and 675mL of dH2O. The 12 samples were incubated at 95⁰C for 10 minutes and the electrophoresis chamber with glass cassettes were assembled with the 1x SDS-PAGE Running Buffer. The 12 samples were loaded into the SDS-PAGE apparatus and ran at 200V for 55 minutes (Kelly et al. 2018).
On lab day 1, a standard BSA curve was produced using a Bradford Assay and a known concentration of BSA protein and the results are displayed in Figure 1 above. The standard BSA curve was produced to help determine the concentration of 6xHis-USP7 on lab day 2. Optical density readings (OD595nm) for various BSA Standards (mg/mL) were recorded using a spectrophotometer at 595nm. The optical density readings increased as the concentration of BSA standard increased. The lowest reading was 0.048OD595nm for 0.025mg/mL of BSA Standard. The highest reading was 0.274OD595nm at 0.400mg/mL of BSA Standard. A blank consisting of 0mg/mL BSA Standard was used to zero the spectrophotometer before taking the first optical density reading. Using a spectrophotometer at 595nm, the concentration of the purified 6xHis-USP7 Elution was then recorded at 0.310OD595nm, on lab day 2. Using the standard BSA curve, the concentration of the purified 6xHis-USP7 Elution was 0.438mg/mL.
On lab day 4, the 12 samples were loaded into a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and ran at 200V for 55 minutes. Two gels were prepared and ran simultaneously. Samples 1 to 6 were loaded and ran in the SDS-PAGE protein purification gel 1 viewed above in Figure 2. Samples 7 to 12 were loaded and ran in the SDS-PAGE GST pull-down gel 2 viewed above in Figure 3. Lane 3 contained the USP7 lysate and Lane 7 contained the dialyzed USP7D in gel 1. USP7D was also loaded into lane 6 in gel 2. Both, USP7 lysate and USP7D, contained two bands at 32kDa and 75kDa. GST peptide resin was loaded into lane 5 in gel 1 and had only one band at 25kDa. GSTI resin containing ICP0 was loaded into lane 6 in gel 1 and only had one band as well at around 32kDa. Eluted GST peptides were loaded into lane 7 in gel 2 and only one band is visible at 25kDa. GSTI Elution which contained both ICP0 and USP7 proteins were loaded into lane 8 in gel 2 and two bands were visible; one band at 32kDa and the other band at 75kDa. It is evident there was non-covalent protein-to-protein interaction between ICP0 and USP7. This is due to the fact there were two bands present in the GSTI Elution, which contained both of the proteins. The band at 75kDa in lane 8 in gel two represents the protein USP7 as the lanes loaded with dialyzed USP7D possessed bands at 75kDa as well. The band at 32kDa in lane 8 represents the protein ICP0 because the GSTI lysate and GSTI resin containing ICP0 loaded in gel 1 both had protein bands at 32kDa. The reason why the GST elution in lane 7 in gel 2 only contained one band that only pertained to the peptides of interest and not USP7 is that USP7 did not possess any protein-to-protein interaction with those proteins and passed through the column when introduced. The GST elution was used as a control.
A standard curve of logMW vs. distance migrated (mm) was produced to calculate the molecular weights for the GST peptides, ICP0, and USP7 and can be viewed above in Figure 4. The USP7 bands migrated farthest distance than the other proteins at 57mm and USP7 had the largest molecular weight of 85kDa. ICP0 migrated at a distance of 42mm and had a molecular weight of 32kDa. GST peptides migrated the least distance of the three proteins at 41mm and had the lowest molecular weight of 30kDa.
The purpose of this experiment was to determine if there were protein-to-protein interactions between the two proteins USP7 and ICP0. The hypothesis was accepted as the two proteins were predicted to have non-covalent protein-to-protein interactions and these results can be supported by literature. ICP0 is a regulatory protein for the Human Herpes simplex virus type 1(Antrobus and Boutell 2008). ICP0 stimulates the reactivation and replication of viral genomes in the latent state of the virus (Antrobus and Boutell 2008). The protein is known to be an ubiquitin ligase and the self-ubiquitination properties ICP0 possesses can lead to the possibility of proteasome degradation that is not beneficial for the virus (Antrobus and Boutell 2008). However, to prevent the proteasome degradation, the protein USP7 is recruited to stabilize ICP0 (Antrobus and Boutell 2008). Human ubiquitin specific protease 7, also known as USP7, removes polyubiquitin chains from ICP0, which prevents proteasome degradation (Pozhidaeva et al. 2015). The protein USP7 is characterized as a deubiquitinating enzyme, which has a role in stabilizing various tumor suppressing and transcription factor proteins (Pozhidaeva et al. 2015). Thus, USP7 is significantly important pertaining to the development of many categorizations of cancer, such as prostate cancer (Pozhidaeva et al. 2015). USP7 possesses five ubiquitin domains, UBL1 to UBL5, where the protein can interact with substrates, such as ICP0 (Pozhidaeva et al. 2015). The substrates interact with these ubiquitin domains in a region known as the USP7C terminal region and ICP0 specifically interacts and binds with the UBL1, UBL2, and UBL3 domains (Pozhidaeva et al. 2015). The findings of this experiment are very significant as many more aspects of the USP7 protein can be explored and studied. The role of USP7’s UBL domains are still greatly unknown and future experiments can be conducted to examine how the several ubiquitin domains located in the C terminus interact with different substrates, such as ICP0 (Pozhidaeva et al. 2015). Further experiments with USP7 examining the ubiquitilating properties the protein possesses will greatly contribute to the future of cancer and antiviral therapies, which will benefit millions of the planet’s human population (Pozhidaeva et al. 2015).
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