Designing a High Affinity Binder to the Viral Protein E6

Designing a High Affinity Binder to the Viral Protein E6

Nakash D Shetty

Degree project in Applied Biotechnology, Masters of Science (45 hp) Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University

Supervisor: Per Jemth

Index:

Abstract………2

1 Introduction…...3

1.1 Cervical cancer………..3

1.2 Human Papilloma Virus………....3

1.3 E6 & PDZ………..4

2 Materials and Methods………...…6

2.1 Mutant PDZ in p RSET A vector………..6

2.2 Touchdown PCR.………..6

2.3 Ligation……….………7

2.4 Expression of mutant PDZ in p RSET A vector………...7

2.5 Purification of mutant PDZ in p RSET A vector………..7

2.6 Mutant PDZ in lipovector……….……….8

2.7 Expression and purification of mutant PDZ in lipo vector………8

2.8 Expression and purification of E6……….……….8

2.9 Stopped flow spectrophotometer………9

2.10 Spectrofluorimeter………..……….10

3 Results………..11

3.1PCR, Digestion, Ligation………...11

3.2 Mutant PDZ in p RSET A vector………...13

3.3 Mutant PDZ in lipovector………..14

3.4 E6 in lipovector………..18

3.5 Binding Experiment………...………20

4 Discussion………...………...22

5 References………...………...23

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Abstract:

Cervical cancer is the second highest cause of cancer related death in women, after breast cancer. The leading cause of cervical cancer is the Human Papilloma Virus (HPV). E6 and E7 are the cancer causing gene products. This project is concentrated on the E6 oncogene of 16 strain of HPV. E6 interacts with the PDZ domain present on various proteins, some of them are tumor suppressors. E6 brings about degradation of the tumor suppressor proteins which leads to cancer. One way to prevent E6 from binding to the PDZ domain is to develop a protein which would bind to the E6 stronger than wild type PDZ. A mutant PDZ domain was obtained by the phage display selection of a mutant library obtained from mutations made on PDZ 2 domain of SAP 97. The mutant PDZ was selected as a high affinity binder to E6. This mutant was expressed in 2TYor terrific medium and purified using immobilized metal ion affinity chromatography and ion exchange chromatography. The protein was confirmed using SDS –PAGE chromatography and mass spectroscopy. E6 was also expressed and purified. Binding studies were carried out between mutant PDZ body and the E6 using stopped flow spectrophotometer and spectrofluorimeter. The results were compared to the binding studies of the wild type PDZ and E6. There was no binding seen between mutant PDZ and E6.

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1 Introduction:

1.1 Cervical cancer

Cervical cancer is the second largest cause of cancer related death in females in the world after breast cancer, more than 12,000 women were diagnosed with cervical cancer in US alone in the year 2008 (1). Every year over 500 million new cases of cervical cancer are diagnosed in the world and over 150,000 deaths are caused by it (2). Cervix is the region which connects the lower part of the uterus to the vagina. The common symptom of this disease is vaginal bleeding and in some cases there could be discharge of vaginal mass, in severe cases it can lead to weight loss, loss of appetite and pelvic pain etc (3). The leading cause of the disease is an infection from the human papillomavirus also known as HPV (4).

1.2 Human Papilloma virus (HPV)

Human papillomavirus belongs to the family of papillomaviridae, these viruses are host specific and are known to cause skin warts. They mostly infect mammals and birds through their keratinocytes i.e. the outermost layer of skin. Structurally these viruses are non-enveloped and they have double stranded DNA as their genetic material which is about 8 kb (5).

HPV is also responsible for various other types of cancer like cancer of the vulva, anal cancer, and penile cancer. Infection of HPV also increases the risk of

oropharyngeal cancer (cancer of the lower part of the throat) (7). There are more than 120 different types of HPV present but only few of them are associated with cancer.

They are described as high risk type for example types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 (8). The majority of the HPV infections are cleared with the human immune system. 70% in the first year itself and 90% in the second year after infection, in the rest 5 to 10% of the cases they can lead to genital warts or develop into cancer (6). Cervical cancer develops when there is regular infection with the high risk type of HPV over a long period of time. Women in developed countries are encouraged to take regular papanicolaou smear test, for this test the cells are drawn from the cervix and the upper region of the uterus to check for the abnormalities. 80%

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of the deaths related to cervical cancer are found in developing countries. In over 90%

of the cases of cervical cancer types 16, 18 and 31 are known to be responsible (4).

HPV infection starts in the basal layer of epidermis, since these cells are in the dividing stage the viral genome is integrated into the genome of the host and utilize the host multiplication mechanism to produce virions. The early viral genes can be seen in the basal stratified epithelial cells which are undergoing cell division. One of the daughter cell which is infected moves away and enters the S phase. The newly synthesized virions reaches the outer layer and they are encapsulated and released outside (9) (17).

The HPV genome consists of 3 parts, a noncoding region, early genes E1, E2, E4, E5, E6, E7 and late genes L1, L2. Early genes are involved in initial viral replication and immortalization of the cells and the late genes are involved in the making of the capsids and release of the virions to the environment. Gene product of E1 has a helicase function, separating the two strands of host of DNA. E2 is involved in the transcription mechanism of the virus and it also acts as a negative control for the oncogenes E6 and E7. E4 comes into action later in the life of the virions, They are involved in the release of the virions into the environment. E5 is known to act upon the killer T cells and thus prevent the degradation of infected basal cells (10). E6 & E7 are classified as oncogenes, since they are involved in maintaining cells at the stage suitable for viral life cycle. They prevent apoptosis and mediate cell progression. E6 &

E7 are also involved in inactivation tumour suppressor genes (11)(12). L1 is involved in the packing of viral DNA into virions, L2 is also involved in this activity. L2 is also known as X2 is involved in the initial integration of viral DNA into host DNA.

1.3 E6 & PDZ

E6 is the oncoprotein which is found in over 90% HPV positive cervical cancer cells.

The E6 protein is about 150 amino acids long. HPV 16 E6 shows a structure of 2 zinc binding motifs, one in the N terminal and one in the C terminal domain. The N terminal zinc binding motif can undergo homodimerization and the C terminal has a tail which interacts with the PDZ domain (16). PDZ domains are 80-90 amino acids long, they are mostly structural domains for signaling complexes in animals, viruses, bacteria and plants (21). PDZ domain interacts with C terminal tail of protein and extends their beta sheet with the help of the binding partner (22). E6 plays a significant role in immortalizing the cells by disrupting telomerase activity and

suppressing the tumor suppressors. E6 in association with E6AP which is an ubiquitin tagging protein brings about degradation of various proteins such as p53 which is a tumor suppressor, notch1 which is a single pass transmembrane protein and various other proteins. The E6-E6AP complex poly- ubiquitinylates the protein, which are then degraded by the 26 S proteasome. E6 has a PDZ binding motif at its C terminal,

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which binds to different PDZ domains which are present in a large number of proteins.

PDZ domain is present in proteins like hscribble which acts like a tumor suppressor in human and MUPP1 down regulation of which is seen in cases of breast cancer. The interaction of E6 with the PDZ domain can lead to loss of cell polarity, some of these proteins are a part of signaling cascade, they also affect cell morphologically. Due to the above mentioned affects it could result in carcinogenesis (4).

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Figure 1: The PDZ2 domain of SAP97 showing the position where the mutations were made, SWISS PDB and VMD viewer was utilized to make this structure. PDB id 2I0L.

The studies conducted by Liu and Gillian show the interaction between PDZ2 domain of SAP97 and the C terminal peptide of HPV 18 E6 (18). To obtain a potential drug against cervical cancer we tried to make a high affinity binder to E6, which would have stronger affinity to E6 compared to the wild type PDZ. PDZ 2 domain of SAP97 was selected for this purpose and mutant library was made by Andreas Karlsson (unpublished). Mutations were made 5 positions on the alpha helix as shown in figure 1, but cysteine was avoided to avoid di-sulphide bridge formation. The mutant library was put through a phage display selection with HPV 16 E6. One mutant survived after three rounds of selection. The mutant PDZ had the following mutations H384A, E385A, V388H, L391F and K392S. This work was done before my arrival to the lab.

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My aim for the thesis was to express this mutant PDZ and to check the binding affinity of this mutant PDZ with the E6.

2 Materials and Methods:

The mutant PDZ was expressed in a vector pComb3 for the preceding phage display method and was called 1R301. We attempted to transfer the mutant in to a high expression vector to be expressed in BL21 strain of E coli. We utilized pRSET A as the high expression vector. pRSET A gives resistance to ampicillin and

chloramphenicol, which would help in selective expression.

2.1 Mutant PDZ in p RSET A vector

The c DNA of the mutant PDZ in pComb3 was present between a SpeI site and BamHI site, so we utilized these two sites to cut mutant PDZ out from pComb3.

pComb3 was amplified overnight in XL1 E coli cells. The plasmid was obtained from the cells with the help of plasmid purification kit by omega biotek and gene jet PCR purification kit was utilized to concentrate it. The purified plasmid was digested with restriction enzymes BamHI and SpeI at 37ºC overnight. The restriction enzymes were inactivated by heating. The digested plasmid was run on the agarose gel and was cut out under UV light, the plasmid was purified from the gel using gel extraction kit by omega biotek.

2.2 Touchdown PCR

The next step was to introduce a SpeI site in pRSET A, which is an expression vector used for high expression of protein in E coli. Touch down PCR was utilized for this method to attain a high level of specificity to the primer. The primer utilized was 36 base pairs long and T M was 60.3º C. In the first stage of annealing there was a decrement from 65 - 50 º C and for the second stage of annealing there was a temperature gradient selected between 50- 60 º C, and the following three

temperatures were selected for annealing 54.1, 56.6, 59.7 º C. The products of PCR were run on 0.5 % agarose gel. Three PCR samples were pooled together, as positive result were seen for all of them on the gel. The remaining of original template DNA was digested with the help of Dpn1at 37 º C. The product from PCR was transformed into E coli XL1 cells and grown overnight. The plasmid purified from transformed XL1 cells were sequenced by Uppsala genome center, 5 out 8 had the SpeI site, so one of them was selected and named as clone 493.

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Clone 493 underwent digestion with BamHI and SpeI, and the digest was run on an agarose gel. The cut plasmid could be seen as a large band between 4000 base pairs and 1000 base pairs, this plasmid was cut out from the gel and purified using gel extraction kit by omega biotek.

2.3 Ligation

The next step in the process was to ligate the cut vector 493 with the PCR fragment from pComb3. There were 4 different molar ratios of the vector to insert tried 1:3, 1:5, 1:7, 1:10. The samples were kept at room temperature overnight and on the next day the T4-ligase was inactivated by heating it. The samples were transformed into E coli XL-1 cells and grown over-night on culture plates. 10 colonies were selected and plasmid was obtained from them, these plasmids were sequenced. Three out of 10 colonies were correct. One of them was selected and denoted as clone 508.

2.4 Expression of mutant PDZ with p RSET A vector

Clone 508 was transformed into E coli BL-21 cells, which were plated on an agar plate with ampicillin and chloramphenicol. One clone was selected from that plate and grown overnight in 50 ml 2TY (terrific broth) media containing ampicillin and

chloramphenicol for the start culture for the expression. 12.5 ml of the start culture was transferred into a flask containing 800 ml of 2TY and ampicillin. The optical density (OD) of the culture is monitored after that. Once the OD reached 0.8 we induced the culture with IPTG and then the culture was grown overnight at 25 ºC.

2.5 Purification of mutant PDZ with p RSET A vector

The next day the cultures were spun down, the supernatant discarded and the pellets were resuspended in 10 ml of nickel binding buffer (50 mM tris/HCl pH 8.5+ 400 mM Nacl + 20 mM imidazole). The pellets were put through ultrasonication to release the protein which is intracellular, and then the sample was ultracentrifuged to separate the cell debris from the lysate. The supernatant was filtered and loaded on the nickel column to purify the protein using the His tag present on the protein. The protein was eluted from the column using 250 mM imidazole.

The protein was dialyzed to get rid of the salt and imidazole in 20 mM tris/HCl pH 8.5 as required for the next step of purification. The membrane used for dialysis is of the size 3500 daltons as the molecular weight of the protein is 11846 daltons.

As a next step of purification the sample was loaded on an anion exchange column ( mono Q column) 20 mM tris was used as loading buffer and 20 mM tris + 1 M Nacl was used for elution. A gradient was made with the elution buffer for eluting the protein. The samples were also checked for absorbance at 280 nm with the help of

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nanodrop to measure the protein concentration. Reversed phase column was employed to concentrate the protein in 1.2 ml of 0.1 % TFA + 60 % acetonitrile.

The protein was confirmed with the help of SDS PAGE and mass spectroscopy. The concentration of the protein obtained was low, so we decided to transfer the gene for mutant PDZ in lipo vector, which expresses a lipoyl domain as a fusion protein.

Previous work in the lab had shown the lipo vector to give a higher expression for other proteins.

2.6 Mutant PDZ in a lipo vector

The vector 290 was the lipo E6 vector which was utilized. This vector tough had a BamHI site, but lacked SpeI site, so alternately an EcoRI site was selected for digestion and ligation of mutant PDZ from our previous clone 508. Insert from clone 508 was digested using BamHI and EcoRI, and ligated into the lipo vector. The product was transformed into XL1 cells and grown overnight on a culture plate. 10 colonies were selected from the plate and plasmids were obtained from them and sequenced. Three out of 10 colonies gave the correct result, so one of them was selected for expression and named as clone 515.

2.7 Expression and purification of mutant PDZ in lipo vector

Clone 515 was expressed in 8 bottles each containing 800 ml of 2TY. Expression was carried out at 37 ºC and the OD was monitored, once OD reached 0.8 the culture flasks were induced with IPTG. The culture was allowed to express for 18 hours at 18 ºC. The protein was purified as mentioned above, the eluate from the nickel column was collected and dialyzed in 50 mM tris/HCl pH 8.5. For dialysis the membrane was used with a molecular weight cut off of 3500 Da, as the protein of interest was

11846.5 Da. The dialysis is an important step as it helps to get rid of imidazole and salt. These would hinder the process of thrombin digestion and would also cause a problem in the ion exchange chromatography which was the next step of purification.

The protein was digested with the help of thrombin for a period of 3 hours to cut out the lipo domain. Thrombin was removed from the mixture by passing it through a high trap benzamidine column by GE Healthcare at a flow rate of 1 ml/min. The next step of purification was to separate out the lipo part of the protein. We utilized the nickel column for this purpose. His tag is attached to the lipo part of the protein, so it is bound to the nickel column. Mutant PDZ is found in the flow through.

For further purification the buffer in which the protein was suspended in must be changed to provide a positive charge to the protein, so the protein was dialyzed against 10 mM potassium phosphate buffer pH 7.0. The protein was purified using cation

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exchange chromatography in 10 mM phosphate buffer pH 7.0 and eluted out by a gradient of elution buffer of 10 mM phosphate buffer with 1 M NaCl. The protein was regularly checked on the SDS-PAGE gel for purity.

2.8 Expression and purification of E6

Protein E6 was also purified for the binding experiments with the mutant PDZ

domain. Clone 239 was the clone used for expression. Clone 239 had E6 with the lipo domain, as E6 could not be expressed on its own, E6 was expressed with the lipo domain attached to it. The expression for E6 was exactly like mutant PDZ domain except all the buffers had to have 2 mM mercaptoethanol, to prevent the formation of di-sulphide bridges. Purification steps would differ compared to the mutant PDZ. The first step of purification involved passing the protein through nickel column so that due to the presence of His tag the protein would bind to the nickel column, and would be eluted out with 250 mM imidazole. The protein is later dialyzed to remove the salt and imidazole. The next step of purification is anion exchange. The buffer used for binding was tris/HCl pH 8.5 with mercaptoethanol and for elution we used the tris/HCl pH 8.5 +1M Nacl with mercaptoethanol. The proteins eluted out were checked using SDS PAGE with E6 protein as the molecular weight standard. The protein was handed over for mass spectroscopy to check the molecular weight.

2.9 Stopped flow spectrophotometer

The next stage of the lab experiments were to check the binding between the mutant PDZ and the E6, for the ease of explanation we would consider a single step reaction A + B ↔ A.B ...eq(1)

The first method employed was the stopped flow method , in this method two solution in this case A and B are placed in different syringes, the components are allowed to mix in a mixing chamber rapidly , detector measures the changes in this system. We utilized an applied photophysics SX-20MV stopped flow spectrometer for

measurement of our binding studies. The parameter used for measuring is the

fluorescence emission, we utilized a light of 280 nm for excitation and measured the emission spectra above 320 nm using a cut off filter. If A has a tryptophan present in its structure, this would be the probe for the experiment, and A is rapidly mixed with B. The binding between A and B would lead to a change in the microenvironment of the tryptophan which would result in the change in emission spectra. The stopped flow method helps us to measure the changes in fluorescence during which the system reaches equilibrium and no further change can be seen. The change in fluorescence is plotted against time to obtain a plot, which can help us determine the rate constant by using a single exponential

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A= A EQ (1-e ^{-kobs t} )+C...eq(2) or a double exponential in case of a biphasic reaction

A= A EQ (1-e ^{-kobs1 t} )+B EQ (1-e ^{-kobs2 t} )+ C...eq(3)

where A is the signal recorded at time t and A _EQ and B _EQ are the amplitudes of the respective phase and k _obs1t and k _obs2 t are the observed rate constants for first and second component of the double exponential reaction

For the stopped flow experiment we used 5μM of the mutant PDZ domain and studied the binding with 10μM of the fusion protein E6 with lipo domain. Excitation was at 280 nm and the emission was measured at >320 nm. The mixtures were measured for duration of 1 sec to 40 sec after mixing. Control measurements were also made of mutant PDZ with the buffer, and the fusion protein E6 with the lipo domain with the buffer. All the experiments were carried out at 10 º C, as these condition were found to be suitable to study the binding of the wild type PDZ with the E6 (19).

2.10 Spectrofluorimeter

Another instrument utilized for the binding experiments was SLM 4800 spectrofluorimeter, this instrument measures the changes in fluorescence over different wavelength and creates an emission spectra. Consider a system in which there is a mixture of component A and B at equilibrium. This mixture is excited by a source of light (280 nm) and the emission is measured in the range x - 400 nm, there will be a wavelength where the intensity of the emission will be the highest. In another scenario when it is just component A and it is exited with the same light and the emission is measured over the same wavelength range, there would be a shift in the wavelength which would give the maximum intensity. This could be explained by the change in the micro-environment of the amino acids when being just A and being in a mixture with B. This shift in the wavelength which gives the maximum intensity could be a test to determine the binding.

The spectrofluorimeter can also be utilized to determine the dissociation coefficient.

Consider reaction 1, we keep the concentration of component A at constant and add component B in continuous increment. We would also monitor the intensity of the emission. We would plot this intensity of the peak against increasing concentration of B and observe a change in the intensity. We would observe in the plot that after a while there won't be any change in the intensity and it would appear a straight line. All the component A would bind to B, the excess of B in the mixture won't make a change

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in the intensity of fluorescence of the system, provided the fluorescence is due to the amino acids in A. With such a graph we would be able to calculate the dissociation coefficient, this graph would be called saturation graph (21).

For our experiments on the spectrofluorimeter 5 μM mutant PDZ and 10 μM C

terminal E6 peptide, the C terminal peptide of E6 is the part of E6 which binds to PDZ domain of SAP 97 (20). We selected the C terminal peptide of E6 over the E6 lipo domain because there were tryptophan in the E6 lipo domain protein, and these residues would have given a background noise. This would make it difficult to see the shift in the wavelength that gives maximum intensity. So a decision was made to go ahead with the C terminal E6 peptide. For the saturation graph mutant PDZ was kept constant at 5 μM and the concentration of E6 peptide was varied from 2.5 μM TO 20 μM. Excitation was at 280 nm and the emission spectra were measured over 300 nm to 430 nm and the saturation data was generated at 355 nm.

3 Results:

3.1 PCR, digestion and ligation

The first major challenge in the project was to insert a SpeI site in pRSET A vector for the ease of introduction of mutant PDZ gene into the high expression vector. We utilized touch down PCR for this step. Touch down PCR is utilized to attain a high level of specificity in the binding of the primer. It is carried out in two steps in the first stage annealing temperature is reduced in decrement, the starting temperature is higher than the melting temperature and it is reduced to more permissible level over 10-15 cycles. In the next stage the cycles are continued in the lowered annealing

temperature. Due to the starting temperature being so high there is a high level of specificity with primer binding, and the correct binding product is amplified in more favourable annealing temperatures (14). Touch down PCR also helps to overcome the setback in cycle conditions and the PCR buffer (13). Figure 2 describes the result of PCR products run on the gel. There can be seen a streak of DNA on the gel,

considering the coiled structure of the plasmid, it runs differently from the straight ladder. Upon sequencing it was confirmed that SpeI site had been introduced in pRSET A (ref figure 2).

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Figure 2: The pcr results of clone pRSET A to introduce the SpeI site before EcorI site. The lane 1 shows the molecular weight ladder lane 2, 3 and 4 shows three different temperature gradients used for the PCR. Lane 5 shows the result of a previous PCR where there was an error in the calculation of T

. The annealing temperature utilized in each lane is as follows Lane 2 is temperate 54.1 º C, lane 3 is temperature 56.6 º C, lane 4 is 59.7 º C and lane 5 is temperature 63.1 º C.

After the SpeI site was successfully inserted into the pRSET A vector, it was digested with BamHI and SpeI restriction enzymes. After successful digestion, the plasmid was run on the agarose gel to separate the insert from the rest of the vector. As seen in figure 3 due to the major difference in the size of the fragment and the remaining vector it was easily able to differentiate and the insert could be cut out and purified.

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Figure 3: The plasmid pRSET A run on a agarose gel after digestion with restriction enzymes BamhHI and SpeI. The length of the plasmid is 3000 bp and the length of the insert after digestion was around 300 bp

Ligation of mutant PDZ into the pRSET A vector was the next step, as we were not sure as to what fragment to vector ratio for ligation would give a good result 4 different ratio of the mutant PDZ to the cut pRSET A plasmid were utilized. The results of ligation were sequenced and the ligation was confirmed.

3.2 Mutant PDZ in p RSET A vector

Expression of clone 508 was carried out at 37ºC and would be induced with IPTG when the OD reaches 0.8. The OD 0.8 is a measure to confirm that all the cells in the culture have grown to the late log phase which is ideal for the expression of the protein. The inducer IPTG binds to the lac repressor and allows the production of protein on the lac operon.

The first step of purification utilizes the property of histidine to bind to the nickel column. The presence of small amount of imidazole in the binding buffer helps in specific binding of the protein. The presence of imidazole ring in the histidine tag attached to the protein helps it to bind to the nickel column, and using a higher concentration of imidazole in the elution buffer helps to elute the protein from the column. The imidazole replaces the histidine bound to the column and the protein

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elutes out. The protein was dialyzed and was put through thrombin digestion to remove the His tag.

The next step of protein separation is charge based separation as described in materials and methods section. We know that the calculated pI of the protein is 8.93 from the prot param tool, by SIB bio informatics tool. The column utilized was anion exchange column namely mono Q column by GE health-care. A gradient created using the elution buffer which is represented by the red line in figure 3. The pH of the buffer utilized for binding was 8.5 and the protein had pI of 8.93, so the protein had a positive charge. The protein eluted out in the flow through, which can be seen as a blue peak which represents high absorbance at 280 nm, before the gradient starts (ref figure 4).

Figure 4: Graph of the anion exchange chromatography of mutant PDZ. The blue line shows the absorbance at 280nm and the red line represents the concentration gradient of the elution buffer. The binding buffer was tris/HCl of pH 8.5 and as the pI of the protein is 8.93 the protein gets eluted out in the flow through.

As the protein concentration was not so high, after anion exchange, reversed phase extraction was utilized to concentrate the protein in a small fraction of 1.2 ml. A conce ntration of 45 μM was achieved and this sample was handed over for mass spectroscopy to derive the exact mass to assure that the mutant protein was expressed correctly.

From the results of mass spectrometry it was confirmed that the protein of interest was present in the flow through of the anion exchange. In an attempt to get a higher

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concentration of mutant PDZ, we tried expressing it with a lipo vector which had given better expression for other proteins expressed previously in the lab.

3.3 Mutant PDZ in lipo vector

The new clone that was formed by introduction of the c DNA of mutant PDZ domain into the lipo vector was denoted clone 515. Clone 515 was initially grown at 37 º C.

Once OD of 0.8 was reached the culture was induced with IPTG and grown at 18 º C for 18 hours. The initial purification was carried out in the same manner as mentioned in materials and methods. A very high absorbance was seen for the product from the nickel column compared to the previous expressions indicating high expression. The mutant protein is expressed with the lipo domain, so to obtain the protein in the purest form it is important to get rid of the lipo domain. The lipo domain is connected to the protein with a linker domain of Leu-val-pro-arg-gly(LVPRG) this is the site for thrombin digestion. We dialyzed the protein overnight as excess of salt is not good for thrombin digestion. The next step after thrombin digestion was to pass it through benzamidine column to remove the thrombin.

The next step in the purification procedure is to get rid of the lipo domain, and for this purpose we utilized nickel column. The His tag is attached to the lipo domain, so the lipo domain stays bound to the column and the protein is found in the flow through.

As a final step we utilized cation exchange column to obtain the protein in the purest form. With the help of prot param tool we know that the mutant PDZ protein has a pI of 8.18, so we decided to use a phosphate buffer (KPi) of pH 7.0 so the protein will have positive charge in this buffer, and is bound to the column. The protein is eluted out with the help of phosphate buffer with 1 M NaCl.

Figure 5: Cation exchange chromatography on an a s column for mutant PDZ protein ( expressed from clone 515) binding buffer was 10mM KPi pH 7.0 and elution buffer 10mM KPi + 1 M Nacl. The blue line is the OD at 280 nm and the green line shows the NaCl gradient 0.0 to 0.5 M in 150 ml by mixing binding buffer and elution buffer.

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Figure 6: The different peaks as separated by the cat ion exchange column as shown in figure 5. Bands 1 to 6 represent the peaks from figure 5 run on a SDS Page chromatography. Band 7 is the original PDZ protein run as reference for comparison.

The gradient utilized for this protocol was 0-50 % of elution buffer in 150 ml, which was a very steep gradient so possibly there could not be a good separation. The separation of the protein can be seen in figure 5 made by measuring OD at 280 nm.

Allot of protein which could not bind to the column is seen in the flowthrough which is represented by peak 1. Peak 2,3,4,5 show the proteins that elute out when the gradient starts for elution buffer, which is represented as a red line in figure 5. The different peaks as it can be seen on figure 5 were collected separately and these peaks were loaded on a gel. A sample of original PDZ protein was loaded on the same gel for molecular weight reference as can be seen in the gel as band number 7 on figure 6.

The peak 2,3,4,5 were the protein which almost travel similar to the original PDZ domain as seen in figure 6, so we decided to pool all the samples and run another gradient of 0-30% of in 120 ml.

Three peaks were obtained from this run as can be seen in figure 7 which were collected separately and loaded on a gel. PDZ domain was also loaded onto the gel as a standard. The results of the run can be seen in figure 8. The peak 2 give a clear band which is similar to the band of PDZ domain , so the peak 2 was dialyzed and handed over for mass spectroscopy for confirming the molecular weight

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Figure 7: Pooling the samples from the previous cation exchange chromatography which are similar to the PDZ domain as seen on the SDS gel. The pooled sample was loaded on another cation exchange column with same binding buffer and elution buffer. The gradient utilized was 0.0 to 0.3 M NaCl in 120 ml buffer.

Figure 8: The three peaks collected separately were loaded on to an SDS PAGE gel. Samples 1,2and 3 represent the peaks from the second cation exchange chromatography and sample 4 is the wild type PDZ which is used as a molecular weight reference. Only the peak 2 shows a clear band similar to the PDZ domain.

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Figure 9: The result of mass spectroscopy analysis of the mutant PDZ domain, The weight was found to be 10567 Da which is similar to the calculated10564 Da. The difference in the molecular weight could be accounted for by the error in mass spectroscopy.

A clear peak can be seen in figure 9 at the molecular weight 10567 dalton which is very similar to the mutant PDZ protein which has a molecular weight 10564 dalton.

So we confirmed that the mutant PDZ domain was obtained in the purest form at a concentration of 37μM in a volume of 12 ml.

3.4 E6 with the lipo vector

E6 was also expressed in a vector with the help of a lipo domain. After the initial purification with the nickel column and dialysis, when the lipo domain was cut out using thrombin the protein precipitated. Unsuccessful attempts were made to dissolve the protein in urea and there was also a risk if the protein would not fold back to its original structure if excess urea was used. A decision was therefore made to express and purify the protein with the lipo domain.

As it was known from previous work on the E6 the pI of the protein was 6.75. For initial purification after the expression, the nickel column was utilized and after that we dialyzed the protein to remove the salt and imidazole. Anion exchange was utilized to further purify the protein, binding buffer was 20 mM tris/HCl + 2 mM

mercaptoethanol pH 8.5, so the protein of interest would have a negative charge and would bind to the column. The elution buffer utilized was 20 mM tris/Hcl +1 M Nacl + 2 mM mercaptoethanol pH 8.5, and the gradient utilized was 0.0 to 0.5 M NaCl in 150 ml achieved by mixing binding buffer and elution buffer.

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Figure 10: E6 with the lipo domain on the anion exchange column. The Binding buffer 20 m M tris/HCl pH 8.5 and elution buffer 20 mMtris/HCl +1M NaCl pH 8.5. All the buffers contained 2 mM mercaptoethanol. The blue line shows OD at 280 nm and the green line represents the gradient of elution buffer 0 to 50% of solution b in 150 ml. All the fractions corresponding to a single peak were collected separately as shown in the figure.

Figure 11: All the peaks from the anion exchange column were collected separately and loaded on the SDS PAGE gel. Lane 1 to 5 represents all the peaks from anion exchange . LANE 6 sample is of previously purified E6 which is the molecular weight reference.

As it can be seen in figure 10 at higher salt concentration there were 3 other protein peaks present. The samples corresponding to different peaks were collected separately and run on the SDS PAGE gel as seen in figure 11. Lipo E6 fusion protein was found only in peak 1. This sample was handed over for mass spectroscopy. The mass of the

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protein was found to be 19652 Da as seen by a clear peak in figure 12, which is very similar to the 19658.2 Da which is the calculated weight of the lipo E6 fusion protein.

The final concentration which was obtained for E6 was 402 μM in a volume of 20 ml.

Figure 12: The mass spectroscopy results of the lipo-E6 after purification. The molecular weight of the protein from mass spectroscopy is 19652 Da and the mass by our calculations is 19658.2 Da which is very similar. Thus we conclude the protein is lipo E6 fusion protein. Noise can be seen in the background that could be due to the presence of mercaptoethanol.

3.5 Binding experiments

The results from the stopped flow spectrometer where the binding was carried out between mutant PDZ and E6 with the lipo domain attached to it, first the binding was checked for 5μM mutant PDZ and 10 μM of the E6. Binding was also checked between mutant PDZ and the buffer. They were checked over the duration of 10 seconds, 20 seconds and 40 seconds. The results from the experiments were compared with the binding studies of PDZ 1354 W with HPV 18 E6 peptide as were performed previously in the lab (ref 20). There were no changes observed in the fluorescence, so it was concluded that there would be no binding between the mutant PDZ domain and the HPV E6 with the lipo attached to it.

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The results from the spectrofluorometric analysis were measured at the equilibrium reached between the mutant PDZ and E6 peptide. The aim of the experiment was to observe the change in the spectra of the binding between the mutant PDZ and E6 and compare it with the spectra of the mutant PDZ in the buffer. The graphs showing the wavelength which gave maximum intensity are shown in figure 13. In an ideal binding there would be a shift in in the wavelength which gives maximum intensity, due to the change in the micro environment of the amino acid which would give the

fluorescence, which is not observed in the figure 13. This provides evidence to show no binding between mutant PDZ and the E6.

Figure 13: Difference in peaks with binding of mutant PDZ with buffer and binding of mutant PDZ with E6.

The next set of experiments to be carried out in the spectrofluorimeter were to obtain a saturation curve by keeping the concentration of PDZ domain constant and increasing the concentration of the C terminal E6 peptide. The results from the saturation curve were compared with the results from the saturation data derived in the lab comparing the PDZ saturation with the C terminal E6 peptide, there was no pattern for saturation observed in the graph as shown in the figure 14, so we could conclude that the C terminal E6 peptide is probably not binding with the mutant PDZ domain.

To conclude the mutant PDZ domain does not have a stronger binding affinity to the E6, compared to the wild type SAP 97 PDZ 2.

wavelength → x axis

fl uros ce nc e→ y axi s

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Figure 14: T he saturation curve by keeping the mutant PDZ constant at 5μM and increasing the conc entration of E6 from 2.5μM to 20 μM in 11 different concentrations. The excitation wavelength was 280 nm and the emission monitored at 355 nm.

4 Discussion:

The first expression of mutant PDZ was carried out in pRSET A vector. The identity of the protein was confirmed with the help of mass spectroscopy. The protein was obtained in the purest form, but the concentration being too low it could not be utilized for further experiments.

To obtain a higher concentration of mutant PDZ the expression was carried out with the lipo vector. Lipo vector was proven to give a high expression for proteins

previously expressed in the lab. Mutant PDZ expressed with the lipo vector gave a protein at a high concentration, which was enough to carry out binding experiments with the E6.

E6 was expressed with the lipo vector. E6 was found to coagulate after the lipo domain was cleaved off. The coagulated E6 was attempted to resuspend with the help of urea, which was not possible. E6 was purified with the lipo domain attached to it.

Mass spectroscopy was utilized to confirm the identity of the protein.

The stopped flow measurements and spectroflurometer experiments were carried out with E6-Lipo fusion protein and C terminal peptide of the E6 protein. The ideal experimental set up would be to utilized the pure E6 protein

The conclusion that can be drawn from the stopped flow measurements and spectroflurometer is that there could be seen no binding between E6-Lipo fusion

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protein and mutant PDZ domain. We were also unable to find out the dissociation constant between mutant PDZ and C terminal tail of E6. In an attempt to find the dissociation constant we could try to draw a saturation curve with using excess of E6 compared to mutant PDZ domain. This would help find the dissociation constant if there is any binding at all in the presence of excess of E6, but it would still fail to find a stronger binder compared to the wild type PDZ.

As an alternative method to check the binding between the mutant PDZ and the E6 we could use gel chromatography and a gel electrophoresis with a non-reducing gel between mutant PDZ and E6. In case there is a binding between the mutant PDZ and the E6, the product would have a greater mass this could be utilized to separate it out from the mixture of mutant PDZ and the E6 which are free in the mixture.

We should also look at the phage display selection process if there could be any other protein during the selection process which bound to the E6 C terminal tail and try to find the dissociation constant with the protein and E6. This could have a stronger binding to E6 than wild type PDZ.

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