Iulia

Niculescu
Investigating

the Interaction Between

Purified
6xHIS-USP7 and GST-EBNA1
213 419 338
Section 10 Bio2070 (Rahima)

13/03/16

MATERIALS AND METHODS

Preparing the Bacteria for Purification
Pre-prepared overnight cultures of bacteria were used to create bacteria with overexpressed
proteins, made possible by adding 45mL of the LB Amp+ Km+ in glutathione S-transferase
(GST) and in 6x Histadine tagged ubiquitin specific processing protease 7 (6xHis-USP7) and
45mL of LB Amp+ in the glutathione S-transferase Epstein-Barr virus nuclear antigen 1 (GSTEBNA1) and incubated for 1 hour at 37C at 250rpm. 550uL of 0.1M of IPTG was added to the
culture and incubated in same conditions. 150uL of the non-induced GST-EBNA1 was added
into a microfuge tube and centrifuged. This was stored at -20C. All other samples were
transferred into empty 50mL tubes.
Purification of 6xHis-USP7 using Ni2+ Affinity chromatography
Lysis/Binding Buffer was created by mixing 5.0mL of Common Buffer and 5.0uL of 5M
Imidazole and adding 1 mL to 6xHis-USP7 which was vortexed and centrifuged. 50uL of Ni2+IMAC agarose resin stock was added to the column with a yellow plug. 1mL of distilled water
(three times) and 1 mL of Lysis/Binding Buffer (three times) were all added to the column
separately, after removing the yellow plug. Samples were centrifuged and when finished, 10uL of
the lysate supernatant (USP7 Lys) was collected and stored at -20C. 1mL of the crude bacterial
cell lysate was also transferred to the column. Buffer and the Elution Buffer were created by
mixing 9.0mL of Common Buffer and 27uL of Imidazole and 5.7mL of Common Buffer and
300uL of Imidazole, respectively. The resin was then incubated. 8mL of Ni2+ Wash Buffer in 1mL
volumes was added and 200uL of Ni2+ Elution Buffer in the column. The final unit was
centrifuged and stored at -20C. The 6xHis-USP7 elution fraction was transferred to a dialysis
tube in the 1xPBS pH 7.4 for 48 hours in 4C. 20uL of dialyzed USP7 was stored away at -20C
once it finished.
Purification of ENBA1 using GST Affinity Chromatography
At the same time, 1 mL of 1xPBS pH 7.4 was added to the bacterial pellet and vortexed and
centrifuged, while a pre-prepared Glutathione-agarose resin was washed with 1mL of distilled
water and 1mL of 1xPBS pH 7.4, each three times. Once the centrifugation was complete,

samples GST Lys and GSTE Lys were collected from the supernatants and stored in -20C.
250uL of this lysate was added to the column, washed with 750uL of 1xPBS pH 7.4, incubated
and centrifuged. 7mL of 1xPBS pH 7.4 was added in 1mL volume intervals and after the last
wash, plugs were attached the the columns so that another wash of 1mL of 1xPBS pH 7.4 could
be suspended and samples could be collected as GST R and GSTE R, stored at -20C.
Determining the Concentration of purified 6xHis-USP7 with Bradford Assay
A Bradford Assay was conducted by mixing 975uL of 1x Bradford Reagent with 25uL of
distilled water, Ni2+ Elution Buffer and 6xHis-USP7 Elt respectively. The spectrophotometer was
set at 595nm and measured each of the samples measure optical density readings. A scatterplot
was then derived to determine the protein concentration of purified 6xHis-USP7.
Preparing samples for GST Pull-Down Assay of USP7 and EBNA1
The columns with GST and GST-EBNA1 were washed again with 1xPBS pH 7.4 and adding
around 200uL of the 6xHis-USP7 and 800uL of 1xPBS pH 7.4 to the GST and EBNA1 loaded
and sealed columns for incubation. The liquid fractions were collected and labelled as GST PDUP and GSTE PD-UP. The columns were then placed on centrifugation and pooled in
collected tubes with the PD-UP samples and stored at -20C. 6mL of 1xPBS pH 7.4 in 1mL
intervals was added to the columns and the final washes were collected, labelled GST-PDW
and GSTE-PDW and stored at -20C. Plugs were attached to the columns and washed with
200uL of GST Elution Buffer. Plugs were removed after an incubation period of 10 minutes and
centrifuged. The elutes were transferred, labelled as GST PD-Elt and GSTE PD-Elt and
20uL of each were stored at -20C.
SDS-PAGE Casting Gels
SDS-PAGE gels made by mixing 50uL of 10% APS to the 5mL of chilled 15% Separating
Solution, along with the addition of 10uL of TEMED. This solution was added to the assembled
glass cassette in the casting apparatus with an overlay of 95% Ethanol and left to polymerize for
25 minutes. Thereafter, the ethanol was decanted and the stacking gel mixture was prepared by
mixing 5uL of food colouring, 25uL of 10% APS, 5uL of TEMED to 2.5mL of Stacking Solution

and added to the glass cassette. It was also left to polymerize for 25 minutes and stored after at
4C.
Casting Gel Electrophoreisis for Prepared Samples
A 1X SDS-Page Running buffer was made from mixing 75mL of the 10x stock to 675mL of
distilled water. To sample 1 (GSTE-N1), 20uL of distilled water, 4uL of 6xPLB and 24uL of Gel
Loading Volume was added. To sample 2 (USP7 Lys) and sample 3 (GSTE Lys), 2uL of 6xPLB
was added. To samples 4 (GST R), 5 (GSTE R), 6 (USP7 D), 7 (GST PD-UP), 8 (GSTE PD-UP),
9 (GST PD-W), 10 (GSTE PD-W), 11 (GST PD-Elt), 12 (GSTE PD-Elt), 4uL of 6x PLB was
added. The protein markers were collected; 42uL of Pre-stained Broad Range Protein Marker and
12uL of BLUeye. These samples were incubated for 10 minutes at 95C. The glass cassette was
removed from storage, the combs were taken out and the gel was submerged in 1X SDS-PAGE
Running Buffer. After the incubation, samples were centrifuged and added to the wells. The gel
was run at 200V for 45 minutes. Once finished, the gels were rinsed with distilled water twice on
the shaker for 5 minutes and submerged with Instant Blue Staining Solution on the shaker for 20
minutes. Samples were then analyzed by taking pictures.

RESULTS

f(x) = 1.1x + 0
R = 0.98

Figure 1, Bradford standard curve plotted on scatterplot with line of best fit with BSA Standard
Concentrations (mg/mL) on x axis and OD Readings at 595nm on y axis, recorded using
spectrophotometry.

Figure 2, Gel #1 image after Ni2+ affinity chromatography on 6xHis-USP7 and GST affinity
chromatography on GST-EBNA1 where lane 1 is empty, lane 2 is the Pre-stained Broad Range
Protein Marker (NEB), lane 3 is the GSTE-NI, lane 4 is USP7-Lys, lane 5 is GSTE-Lys, lane 6 is
GST-R, lane 7 and 8 is GSTE-R, lane 9 is USP7-D and lane 10 is marker BLUeye. Affinity
chromatographies were performed with SDS-PAGE gel electrophoresis at 200V for 45 minutes
on a 15% casting gel and 4% stacking gel, picture taken by camera.

Figure 3, Gel #2 after protein pull-down assay was complete between GST-EBNA1 and 6xHisUSP7, where lane 1 is empty, lane 2 is the pre-stained broad range marker (NEB), lane 3 is GST
PD-UP, lane 4 is the GSTE PD-UP, lane 5 is the GST PD-W, lane 6 is the GSTE PP-W, lane 7 is
the GST PD Elt, lane 8 is the GSTE PD Elt, lane 9 is the Blueye marker and lane 10 is empty.
Affinity chromatographies were performed with SDS-PAGE gel electrophoresis at 200V for 45
minutes on a 15% casting gel and 4% stacking gel, picture taken by camera.

f(x) = - 0.27x + 2.31

R = 0.89

Figure 4, scatterplot with line of best fit (y = -0.2696x + 2.3124) showing the relationship
between distance travelled by protein fragments (cm) in the 15% casting gel by gel
electrophoresis and the log of the molecular weight of respective proteins (logkDa) determined
by cross-referencing the molecular marker with the travelled proteins.
Table 2, Experimentally determined molecular weights calculated from line of best fit
Protein

Distanced travelled
(cm)

USP7
GST-R
GST-EBNA1

3.30
3.65
3.70

Molecular weight
experimentally
determined (kDa)
26.6
21.3
20.6

Molecular weights
(kDa)
25
26
28

RESULTS
In figure 2, The Ni2+ affinity chromatography and GST affinity gel electrophoreses conducted
yielded protein movement in a total of 6 lanes. The first lane was empty. The second lane shows
the Pre-Stained Broad Range Protein Marker (NEB) ladder. The third is the GSTE-NI (noninduced GST-EBNA1), the fourth is the USP7-Lys and the fifth is the GSTE-Lys (lysates). These
three lanes show denaturation of the protein and travelling fragments. Darker fragments are
observed at the 25kDa mark in all three lanes, as well as one thick, dark band slightly higher than
25kDa (around 27kDa) in lane five. The sixth lane is the GST-R (GST resin), the seventh and
eighth lane is GSTE-R (GST-EBNA1 resin), the ninth is USP7-D (USP7 dialysis). These lanes
show minimal to no streaking and only one fragment in each lane. In lane 6, one thin fragment is
detected at 25kDa. It is noted that the seventh and eighth lane having the same protein repeated
was a mistake, and as a result filled in one more lane that was needed. In both lanes, one
fragment is detected at around 28kDa. In lane 9, there were no detected fragments. The tenth lane
is the final ladder, BLUeye Prestained Protein ladder.
In figure 3, the second gel is the pull-down assay between proteins GST-EBNA1 and 6xHisUSP7. The first lane is empty and the second shows the NEB ladder. Lane 3 is the GST PD-UP
(GST pull down unbound proteins), lane 4 is the GSTE PD-UP (GST-ENBA1 pull down
unbound proteins), lane 5 is the GST PD-W (GST pull down wash), lane 6 is the GSTE PD-W
(GST-EBNA1 Pull down wash) and lane 7 is the GST PD Elt (pull down elution). These lanes
show no present fragments or smearing of the protein. Only lane 8 (GSTE PD Elt) shows two
clear, separate fragments at 25kDa and 30kDa. Lane 10 is the BLUeye marker.
In figure 1, the Bradford standard curve was plotted, achieved by plotting BSA Standard
Concentrations (mg/mL) over the experimentally determined OD readings from the
spectrophotometer at 595nm. The standard curve shows a slope of 1.0965 and an equation of the
line of best fit being y = 1.0965x + 0.0021 and a R2 value of 0.98458.
In figure 4, the standard curve of the distance travelled by the protein in cm is plotted against the
log of molecular weight of protein in kDa on a scatterplot. This was determined by physically
measuring the distances travelled by the various samples on the affinity gel electrophoreses

starting from the top of the gel to the fragments and observing the molecular weights of each
fragment. This relationship yielded a slope of -0.2696, a line of best fit of y = -0.2696x + 2.3124
and a R2 value of 0.89039.
DISCUSSION:
Ubiquitin is an essential protein in eukaryotic cells as it functions as a tag to signal in
degradation of specific proteins it is attached to. On the other hand, USP7 (ubiquitin specific
protease) removes ubiquitin from its substrates, to stabilize the bound protein. As a result, USP7
is suggested to have tumour suppressing enzyme activity namely on protein p53, which plays a
vital role in tumor suppression as it can initiate events such as cell apoptosis (Li et al. 2004). The
enzyme activity of USP7 protects p53 from degradation and as a result, will not allow tumors to
proliferate. However, the activity of yet another protein called EBNA1 (Ebstein-Barr virus) is
responsible for some forms of cancer as its expression is present in EBV tumours and it is an
initiator for gene transcription. In addition, it has been shown that USP7 can interact with
EBNA1 and shows affinity to it. This affinity impacts the two proteins in a way so that EBNA1
must compete with p53 to bind to USP7 (Saridakis et al. 2005). Keeping in line with this theme,
this experiment sought out to seek whether or not there is an interaction between USP7 and
EBNA1 through affinity chromatographies and pull-down assays.
The first experimental results determined were from Ni2+ affinity chromatography on the 6xHisUSP7 and the GST affinity chromatography on GST-EBNA1 in the first gel, obtained by SDSPAGE (sodium dodecyl sulfate polyacrylamide gel electrophoreses). Proteins coated by SDS
become negatively charged and denatured. Once under an electric voltage, the proteins move to a
positive end thus allowing for the detection of fragments (Rath et al. 2013). Various samples
were inserted in the lanes to undergo electrophoresis consisting of two ladders used as markers in
order to verify the protein weights of the travelling fragments in lanes 2 and 10.
A number of interactions were seen in each sample. In each affinity chromatography, Ni2+ metal
ion was bound to USP7 and GST and GSTE were first bounded to reduced gluthathione (GSH)
(Kelly et al. 2016). Lane 3 shows the GSTE-NI which the non-induced GST-ENA1, where its
denaturation and protein composition is shown. Since it had not been purified, its lane shows

many fragments, just as lanes 4 (for USP7-Lys) and 5 (GSTE-Lys) show multiple fragments as
well, due to the fact that the lysates which were washed with Ni2+ lysis/binding buffer were not
yet purified. Observing lanes 6 (GST-R), 7 (GSTE-R) and 8 (GSTE-R), there is only one
fragment in each, meaning that the protein had been purified. In lane 6 and 7, the GST-R and
GSTE-R is present as the suspended resin that was present in columns after purification. The
presence of their purified fragments suggests that it is possible to determine their molecular
weight by using the marker ladder. GST-R has a molecular weight of around 25kDa, while
GSTE-R has a weight of around 28kDa. In lane 8, there was an error in the experiment where the
insertion of sample GSTE-R was repeated, and as a result created an identical fragment copy
from lane 7. In lane 8, no fragments for USP7-D are detected. The dialysis of USP7 was done in
order to remove the smaller proteins and purify the eluted fraction. Because of this, there may
have been an error in its purification since no fragment was detected. The movement of the
fragments are dependent upon their charge and size, as well the concentration of the
polyacrylamide gel. If the concentration of the gel is higher, the pores in the gel will be smaller,
so only smaller proteins may pass through the gel, while a lower concentration will cause pores
to be greater allowing for more proteins to pass (Rath et al. 2013). For this particular experiment,
two 15% casting gels were used composed of the 4% stacking gel (for a better resolution) was
overlaid on the 15% gel.
Gel #2 determines the interaction between GST-EBNA1 and 6xHis-USP7 by generating two
fragments in the pull-down assay. One fragment is located around 25kDa and the other fragment
is located slightly above it around 28kDa. It is known that GSTE-EBNA1 molecular weight is
28kDa and 6xHis-USP7 molecular weight is 25kDa, suggesting that both these proteins appear
together in lane 8 (Kelly et al. 2016). Lane 8 is the GSTE PD-UP lane where purified and
dialyzed 6xHis-USP7 was added to the loaded columns containing GST-EBNA1. After being
washed by 1X PBS pH 7.4, the columns contained the two unbound proteins and the presence of
both fragments suggests that one protein was successful at binding the other protein in the resin,
outlining the interaction between the two (Kelly et al. 2015). This conclusion of positive protein
interaction is in line with other research papers as well, where USP7 was found to interact with
EBNA1 in epithelial and B cells. Data from this research shows structurally as well how EBNA1
interferes with the binding of USP7 to p53. (Saridakis et al. 2005).

During this experiment, the concentration of purified 6xHis-USP7 was also experimentally
determined by Bradford Assay where a standard curve was plotted (Figure 1) with a line of best
fit. The line of best fit y= 1.0965x + 0.0021 was used further to substitute the OD reading at
595nm of 6xHis-USP7 Elt as x to determine the concentration. As a result, the concentration is
0.358mg/mL by substituting the reading of OD595nm = 0.325. Its important to note that the R2
value for this line of best fit is 0.98 which is fairly close to 1 and can be interpreted as fairly
accurate.
The molecular weight standard curve was also plotted to determine the relationship between the
molecular weight (kDa) and the distance travelled by proteins (cm). Measurements were taken
off the gels starting from the top to the bottom and molecular weights were observed. These
weights were transformed logarithmically and plotted against the distances travelled. As a result,
a negative correlation was observed between the two. As the molecular weight decreases, the
distance travelled by the protein increases. This is due to the fact that lighter proteins tend to
travel further (Lodish H, Berk A, Zipursky SL, et al. 2000). The slope of the line (y = -0.2696x +
2.3124) can be used to theoretically determine molecular weights as well. For example,
substituting x= 3.30cm, (the distance travelled by the USP7 fragment) a theoretical value of 26.6
kDa is determined, which is close to that of the known value, 25kDa. Values tend to differ for the
other proteins and this can be explained by the correlation value of R2=0.89 which is slightly far
off from 1, suggesting it is not as accurate. This is shown in Table 2.
The data from this experiment concludes that there is an interaction between 6xHis-USP7 and
GST-EBNA1. Through Ni2+ and GST affinity chromatography, the two proteins were purified for
future pull-down assay where their presence at their known molecular weights proved their
interaction. This knowledge is ground-breaking as future cancer studies can delve deeper into
research on EBNA1 and its deactivation or the activation of USP7 and pave the way for
prevention of tumors.

REFERENCES:
Kelly, T. (2016) Research Methods in Cell and Molecular Biology. Department of Biology. York
University. Pp104-110
Lodish H, Berk A, Zipursky SL, et al. (2000) Molecular Cell Biology. New York: W.H. Freeman.
4th ed, section 3.5
Li M, Brooks C, Kon N, Gu W. (2004) A Dynamic Role of HAUSP in the p53-Mdm2 Pathway.
Molecular Cell. 6(13), pp879-886.
Rath A, Cunningham F, Deber CM. (2013) Acrylamide concentration determines the direction
and magnitude of helical membrane protein gel shifts. PMC, 110(39), pp15668-15673
Saridakis V, Sheng Y, Sarkari F, Holowaty M, Shire K, Nyugen T, Zhang R, Lioa J, Lee W,
Edwards A, Arrowsmith C, Frappier L. (2005) Structue of the p53 Binding Domain of
HAUSP/USP7 Bound to Epstein-Barr Nuclear Antigen 1. Molecular Cell. 1(18), p25-36.