Human papillomavirus 45 oncoprotein E7 and Cyclin-dependent kinase interacting protein p21
Over expression, purification and biophysical characterization
Aravindan Varadarajan
Degree project inapplied biotechnology, Master ofScience (2years), 2011 Examensarbete itillämpad bioteknik 45 hp tillmasterexamen, 2011
Biology Education Centre and Department ofMedical Biochemistry and Microbiology, Uppsala University
Supervisor: Associate Prof. Per Jemth
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TABLE OF CONTENTS
1. SUMMARY ...4
2. ABBREVIATIONS ...5
3. INTRODUCTION ... 6
3.1 Structure and Function of HPV E7 ... 7
3.2 E7 interactions with pRb ... 7
3.3 E7 interactions with p21CIP1 ... 8
4. AIM ... 10
5. RESULTS ... 11
5.1 Cloning ... 11
5.2 Overexpression and Purification of His-tagged 45E7- F ... 12
5.3 Refolding and stability studies with E7 ... 13
5.4 Equilibrium denaturation experiment with E7 ... 13
5.5 Refolding and Binding studies with E7 ... 14
5.6 Overexpression and Purification of His-tagged p21CIP1-C ... 14
5.7 Equilibrium denaturation experiment with p21CIP1-C ... 15
6. DISCUSSION ... 17
7. CONCLUSION ... 19
8. MATERIALS AND METHODS ... 20
8.1 Description of constructs used ... 20
8.2 Description of media used ... 20
8.3 Description of E.coli cells used for cloning and over expression ... 20
8.4 Cloning protocol to generate 45 E7 and p21CIP1-C expression constructs ... 20
8.5 Protein over expression ... 22
8.6 Protein purification... 22
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8.7 Purification of E7 ... 22
8.8 Purification of p21CIP1-C ... 23
8.9 Stability experiments ... 24
9. ACKNOWLEDGEMENTS ... 25
10. REFERENCES ... 26
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1. SUMMARY
Human papilloma virus (HPV) is one of the most common sexually transmitted virus groups that cause cervical cancer in women. Based on their pathogenicity, HPV genotypes were classified as high risk and low risk types. E6 and E7 are vital proteins expressed by high risk HPVs were considered as forward players involved in the pathogenesis of cervical cancer. E7 interacts with pRb protein, a transcription regulation factor of the E2F family and p21CIP1 protein, a member of the CIP/KIP family of CDKIs, leading to cell transformation. Recent biophysical studies have shown that full length E7 protein from HPV high risk type 45 has a highly unstructured N- terminal and a structured C-terminal region and presented the first structure of the dimeric oncoprotein.
The aim of this study was to optimize the expression and purification conditions of HPV 45E7 and p21CIP1C terminal peptide and to determine their binding kinetics. Therefore, E7 and p21CIP1- C terminal peptide were cloned, expressed and purified successfully and the purity was >90% as analyzed on SDS-PAGE gel.
Equilibrium denaturation experiments were carried out to determine the stability of these proteins. These results determined that E7 was highly stable and showed no significant changes in the free energies upon unfolding. Furthermore, p21CIP1-C showed linear changes in their free energies upon unfolding, indicating that it remained in the native, unfolded form. When expressed and purified again to perform binding experiments, E7 was apparently precipitated during dialysis in the presence of Zn and DTT. Therefore, it was concluded that DTT should be avoided as a reducing agent for Zn dependent proteins.
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2. ABBREVIATIONS
HPV Human papilloma virus
pRb Protein retinoblastoma
p21CIP1 Cdk-interacting protein
p21 CIP1-C C terminal of Cdk-interacting protein
CDKI Cyclin-Dependent Kinase Inhibitor
PCNA Proliferating Cell Nuclear Antigen
Zn Zinc
DTT Dithiothreitol
SDS Sodium dodecyl sulfate
IMAC Immobilized Metal Affinity Chromatography
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
PCR Polymerase chain reaction
DMSO Dimethyl sulfoxide
IPTG Isopropyl β-D thiogalactopyranoside
Tris 2-Amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride
EDTA Ethylene diamine tetra acetic acid
TBE buffer Tris- borate, EDTA).
TFA Trifluoroacetic acid
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3. INTRODUCTION
Human papilloma virus (HPV) is a group of viruses that can cause several diseases in humans ranging from hand warts, feet warts, genital warts and cervical cancer in women and penile cancer in men. There are more than 200 different types of known HPVs at present and 100 were fully studied at molecular level and were classified as high risk and low risk papillomas (Munger et al. 2004). Among the mucosa-infecting HPV types, high risk HPV 16, 18, 31, 33 and 45 are causative agents for malignancies leading to cervical cancer and low risk HPV 6 and 11 cause benign genital warts (zur Hausen, 1996; Villa, 1997; Bosch and de Sanjose, 2003).
The HPV genome encodes 9 proteins that include the E6 and E7 oncoproteins (Figure 1). The consistent expression of E6 and E7 in cervical tumors is the main cause of carcinogenesis (Munger et al. 2004). Together, these two proteins are good enough to immortalize normal human cells.
E6 interacts with p53, forcing it to a proteasome-mediated degradation process. E7 interacts with a large number of diverse cellular proteins, such as TATA-binding proteins, pyruvate kinase M2, DnaJ and with two most important proteins at the C-terminal domain, retinoblastoma (pRb) and Cdk-interacting protein (p21CIP1) involved in cell cycle regulation and apoptosis.
Figure 1. The HPV genome (7904 bp) is shown as a black circle in middle. The six proteins [E1, E2, E4, E5, E6 (in purple) and E7 (in blue)] expressed at different stages during epithelial cell transformation are indicated in different colors. The specific activity of each protein is indicated with a black arrow.
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3.1 Structure and Function of HPV E7
HPV E7 is a small acidic protein that consists of about 100 amino acids. It has a highly conserved unstructured N-terminal domain (CR1 & CR2) (Figure 2), which share some sequence and functional homology with adenovirus E1A protein and the SV40 large T-antigen (Phelps et al. 1992). The CR-2 region consists of solvent exposed LXCXE motif (aa 22-26) that mediates the binding of B-box of retinoblastoma tumor suppressor (pRb) (Barbosa et al.1990; Lee et al.
1998; Singh et al. 2005). The C-terminal (CR3) region is well structured with β1β2α1β3α2 topology (Figure 3). It consists of two CXXC zinc-binding motifs separated by 29 amino acids (Figure 2), which is responsible for zinc dependent dimerization and mediates inhibition of various cellular ligands at different domains, most specifically cell cycle progression regulator pRb and cyclin-dependent kinase inhibitor p21 and p27 proteins (Zwerschke and Jansen-Durr, 2000; Munger et al., 2001).
pRb binding domain
Dimerization domain
Figure 2. HPV 45E7 full length sequence showing (CR1&CR2) N-terminal region and (CR3) C- terminal region with 2 CXXC motifs.
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Figure 3. NMR structure of the C-terminal domain (dimer) of HPV45 oncoprotein E7. PDB ID- 2F8B. Helixes are shown in rose and sheets are shown in yellow. Zinc ions are indicated as blue spheres
3.2 E7 interactions with pRb
pRb is a tumor suppressor protein that belongs to a family of related proteins such as p107 and p130 (Sidle et al. 1996). pRb binds E2F transcription factors and blocks their transcriptional activation function (Liu et al. 2006). Binding of E7 with pRb dissociates E2F transcription factor from the pRb-E2F complex leading to the transcriptional activation of S phase genes (Harbour et al. 2004).
pRb consists of 3 domains specified as A,B and C. The A and B domain have the high affinity to the E2F transcription factor. The C domain rather show very low affinity and interacts with E7.
The binding of E7 with pRb C-terminal disrupts the E2F binding at the N-terminal site, leading to the dissociation of E2F and facilitation of cells transformation.
3.3 E7 interactions with p21
CIP1p21CIP1 is the member of the CIP/KIP family of cdk-inhibitory proteins, including p21 CIP, p21 KIP1, p27 KIP2 and p21 XIC1 that shares sequence homology in their N-terminal regions. This highly conserved N-terminal region is sufficient to inhibit the Cyclin-cdks (Gulbis et al. 1996).
The C-terminal region of p21CIP1 (GRKRRQTSMTDFYHSKRRLIFSKRKP) has a binding site
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for E7, PCNA and several other cellular proteins. The N-terminal part undergoes a dramatic disorder-to-order transition upon binding to CDK2 (Kriwacki et al., 1996). The C-terminal part of p21 CIP1 refolds into a stable conformation upon binding to PCNA (Gulbis et al., 1996; Funk et al., 1997; Jones et al., 1997; Dotto, 2000; Helt et al., 2002). The key function of p21 is to interact with PCNA and stop the DNA replication process by binding DNA polymerase as a whole complex (Chen et al. 1996; Pan et al. 1995; Warbrick et al. 1995). Binding of E7 with p21 blocks its interaction with PCNA, this in turn blocks PCNA-p21CIP1 interaction with DNA polymerase leading to uncontrolled DNA replication process (Jo Beth Harry et al. 1997).
Earlier biophysical studies have determined the structure of dimeric oncoprotein HPV 45E7 and mapped the residues that bind the C-terminal region of p21CIP1 (Ohlenschläger et al. 2006). To further understand the interaction pattern and kinetic mechanism, these proteins should be expressed and purified first in sufficient amount with good purity. Therefore, we have reported the expression and purification procedure for E7 and p21CIP1. In addition, we have determined the stability of these proteins by performing denaturation experiments.
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4. AIM
The aim of this study was to optimize the expression and purification conditions of HPV 45E7 and p21CIP1-C terminal peptide and to determine their kinetic binding mechanism. These studies would provide a kinetic-based insight in to the complex interaction properties of E7 and would provide a base line for designing inhibitors to block E7 interactions with other proteins.
The project was divided into 3 parts
Part 1: Cloning, Expression and Purification of E7 Part 2: Cloning, Expression and Purification of p21CIP1-C Part 3: Biophysical characterization of E7 and p21CIP1-C
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5. RESULTS
5.1 Cloning
HPV 45E7 full length, HPV 16E7 full length, HPV 16E7-C terminal and HPV 16E7 C-terminal genes were amplified by PCR reaction (Figure 4) and cloned using the pRSET construct.
p21CIP1-C terminal and p21CIP1 full-length genes were amplified by PCR reaction (Figure 5) and cloned in to pRSET construct with a fusion lipo-tag containing a thrombin site. In this project the 45E7-F and p21CIP1-C constructs were used for the over expression.
1 2 3 4
Figure 4. Agarose gel of PCR analysis to verify the presence of full-length HPV 45E7, HPV 16E7 and HPV 16E7-Cterminal.
Lanes: 1. DNA size marker, 2. 45E7-F, 3.
16E7-F, 4. 16 E7-C.
1 2 3 4
Figure 5. Agarose gel of PCR analysis to verify the presence of full-length p21CIP1 and p21CIP1- C-terminal.
Lanes: 1. DNA size marker, 2. p21CIP1-C, 3.p21CIP1-C, 4. 16 p21CIP1-F.
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5.2 Overexpression and Purification of His-tagged 45E7- F
The expression of recombinant protein in E. coli BL21 (DE3) cells resulted in high expression of E7. Less pure E7 was obtained after initial IMAC purification. Therefore, this step was followed by anion exchange chromatography which resulted in better separation of E7. Peak fractions ran on SDS-PAGE showed an additional band larger than E7 indicating the possible presence of E7 in a dimer form. The aim was to obtain pure E7 in a monomeric form. Therefore, purification was continued with reversed phase chromatography to obtain pure E7 monomer. Peak fractions eluted after this step was ran on SDS-PAGE to confirm the purity. Even after this step the E7 was not pure, few more additional bands appeared on the SDS-PAGE gel. Hence, the sample was lyophilized and loaded on to an anion exchange (Q) column which resulted in highly pure E7.
The purity was confirmed to be >90% by mass spectroscopy and SDS-PAGE (Figure 6). The final concentration of the protein was estimated to be 29 µM. Subsequently the protein was unfolded in 8M urea
1 2
Figure 6. Over expressed and purified HPV 45E7-F.
SDS PAGE Lanes: 1. PDZ domain (10kd), 2. E7 (12.5kd) fraction eluted from reverse phase column
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5.3 Refolding and stability studies with E7
To perform biophysical studies, the purified protein was subjected to refolding. Previous studies have determined that zinc is necessary for the proper folding and dimerization of E7 (Ohlenschläger et al. 2006). In contrast, earlier studies have also shown that zinc is not necessary for refolding, since strong cystine bound zinc is present right from the early folding step in cytosol and it is present even in the urea unfolded E7 (Alonso et al. 2002). Therefore, both conditions were tried separately by dialysing urea denatured E7 against 50 mM KPI pH 7.5, 2 mM mercaptoethanol with 5 mM ZnSO4 and without ZnSO4.
5.4 Equilibrium denaturation experiment with E7
Equilibrium denaturation experiment for E7 was carried out in 50 mM phosphate buffer pH 7.5.
This estimated the stability of E7 both in the presence and absence of ZnSO4. Fluorescence intensity values generated by spectrofluorimetry were plotted against urea concentration and fitted to solvent denaturation of a two-state protein equation (Fersht 1999) (Figure 7). The values for mD-N(Slope) and [D] 50% - midpoint (Urea concentration) were generated from the equation and ∆GD-N in water was calculated, with free fitted mD-Nparameters (Table 1) These results apparently showed a right shift in the midpoint value. Therefore, it could be concluded that, E7 remained in a more stable form upon urea denaturation.
Figure 7. Urea denaturation of HPV 45E7-F. Blue line indicates the denaturation cure in the presence of zinc. Red line indicated the denaturation cure in the absence of zinc. X-axis specifies the concentrations of urea in molar and Y-axis specifies the fluorescence intensity in nanometers
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Table 1. Free-fitted parameters obtained from equilibrium denaturation experiments
Protein
mD-N value.
Free-fitted kcal mol-1M-1
[Urea]50%
M
ΔGD-NH2O kcal/mol
HPV 45E7-F with ZnSO4
1.01 4.02 4.10
HPV 45E7-F without ZnSO4
0.64 2.86 1.84
5.5 Refolding and Binding studies with E7
E7 was expressed and purified again to perform binding experiments with p21CIP1-C peptide.
This time the refolding was done using a different method. Previous studies have shown that E7 can be refolded by dialyzing the sample with 50 volumes of 5 mM Hepes (pH 7.2), 5% glycerol, 1 mM DTT and 20 µM Zn-acetate (Braspenning et al. 1997). Therefore this procedure was followed assuming that the protein would be refolded completely. Unfortunately the above process was totally failed due to the heavy loss of protein during the dialysis process. The amount of protein present before dialysis was approximately 10 mg and after dialysis it was less than 1mg. To further confirm this, sample before and after dialysis was run on SDS-PAGE. SDS gel showed a strong band for the undialysed sample, indicating the presence of pure denatured protein and a faint band for the dialysed sample indicating the presence of very small amounts of protein after dialysis
5.6 Overexpression and Purification of His-tagged p21
CIP1-C
Over expression of the recombinant peptide p21CIP1-C in E. coli BL21 (DE3) cells resulted in high expression but with less purity after the initial IMAC purification. Therefore, this step was followed by anion-exchange chromatography which resulted in better separation of the fusion peptide. The fusion tag was successfully cleaved by thrombin digestion. Again, purifying the digested sample on IMAC column resulted in complete separation of peptide from the mixture.
The purification was continued with reversed phase chromatography, which resulted in pure p21CIP1-C peptide. The purity was confirmed to be >90% by mass spectroscopy and SDS-PAGE (Figure 8). The final concentration of the protein was estimated to be 97 µM.
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1 2 3 4 5 6 7
Figure 8. Over expressed and purified p21CIP1-C
SDS-PAGE Lanes: 1. Tandem protein (26kd) 2. Uncleaved p21CIP1-C (14 kd) 3. Cleaved p21CIP1-C (3.4kd) after digestion, 4-6. Flow through fractions containing pure p21CIP1-C (2.5 kd) after IMAC purification. 7. Lipo tag (10 kd) eluted separately from the IMAC column
5.7 Equilibrium denaturation experiment with p21
CIP1-C
Equilibrium denaturation experiment for p21CIP1-C was carried out in 50 mM phosphate buffer, pH 7.5. Fluorescence intensity values generated by spectrofluorimetry were plotted against urea concentration and fitted to solvent denaturation of a two-state protein equation (Fersht 1999) (Figure 9). These results showed no change in the free energy upon unfolding. This showed that, perhaps p21CIP1-C short peptide remained in the native, unfolded form both in 8 M and 0 M urea.
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Figure 9. Urea denaturation of p21CIP1. X-axis specifies the concentrations of urea in molar and Y-axis specifies the fluorescence intensity in nanometers
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6. DISCUSSION
I have cloned the E7 gene of cervical cancer-associated HPV45 and p21CIP1-C terminal peptide into E. coli and optimize the expression and purification conditions to obtain the recombinant protein in sufficient quantity with good purity. Here I discuss the expression and purification procedure to obtain >90% pure E7 and p21CIP1-C protein in sufficient amounts needed for biophysical characterization.
The expression of E7 was high when the E.coli cells were induced over night at 18°C. The purification scheme that I have developed combines three different chromatography procedures.
The IMAC column proved to be of particular value since it separated E7 from most of the E.coli proteins. The second step, anion–exchange purification provided better purity since it separates the protein according to their negative charge. The third step, reversed phase purification further enhanced the purity of E7 since it separates proteins based on hydrophobicity. Finally, anion- exchange coloum was used again which provided E7 with the purity of >90%.
Cellular environment play a vital role in maintaining the stable 3-Dimensional structure of a protein. Any physical or chemical changes in the environment can change the structural and functional properties of proteins. It is widely accepted that proteins can be completely unfolded in aqueous solution by increasing the concentrations of chemical reagents like urea and guanidine hydrochloride (Fersht 1999). Therefore conformational stability of any wild type and mutant proteins can be studied by denaturing it in the presence urea or guanidine hydrochloride (Myers 1996). Stability experiment carried out with E7 showed no significant difference in the free energies upon step-wise urea unfolding, both in the presence and the absence of Zn.
Therefore it may be concluded that, E7 remained in a stable state even in the denaturing conditions.
Earlier studies on metal dependent proteins showed the negative effect of DDT on protein stability (Fersht el al. 1997). It was determined that, DDT molecule affect the stability of zinc dependent proteins by acting as a metal scavenger rather than a reducing agent, forming a stable insoluble DDT-zinc complex (Cornell NW & Crivaro KE 1972) and also the usage of buffers containing DTT has made the protein to disappear virtually (Alonso et al. 2002). Therefore, the possible explanation for the heavy loss of protein after dialysis might be due to the formation of insoluble DDT-zinc complex, which could have eventually removed the zinc from the E7 and made the maximum amount of protein insoluble. Hence, based on the previous studies and the results obtained from our study, it can be concluded that, mercaptoethanol can be used as a reducing for Zn dependent proteins instead of DTT or DTT can be used alone in the absence of Zinc.
The expression level of p21CIP1-C was high when the E.coli cells were induced over night at 18°C. The initial IMAC column separated almost 80% of E.coli proteins in a single step.
Anion–exchange purification provided better purity since it separates the protein according to their negative charge. The fusion tag was cleaved by thrombin digestion. The second IMAC
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purification separated the peptide from the fusion tag. The final step, reversed phase purification further enhanced the separation and provided p21CIP1-C with the purity of >90%
Stability measurements with p21CIP1-C showed no significant difference in the free energies upon step-wise urea unfolding. This might be because of a single tyrosin present on the surface of the peptide or may be the whole peptide remained in a native unfolded state. This should be further confirmed by performing CD experiment, which can provide supplementary information about the secondary structure formation of p21CIP1-C.
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7. CONCLUSION
Based on the above results obtained from numerous trails, it was possible for us to optimize the conditions for expression and purification of 45E7-F and p21CIP1-C. Therefore, this protocol can be used to obtain these proteins in sufficient amount with high purity. In addition, the denaturation studies apparently provided some valuable information about the stability of these proteins. To understand the interaction kinetics of E7 with p21CIP1-C, binding experiments should be carried out using stopped flow, which would eventually draw a baseline to design specific inhibitors for blocking such interactions.
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8. MATERIALS AND METHODS
8.1 Description of constructs used
The HPV 45E7 gene was amplified from HPV cDNA library by PCR reaction and cloned in to pRSET construct, which contains a His-tag followed by additional (LVPRGS) residues at N- terminal for overexpression and purification purpose. p21-C gene was amplified from cDNA library by PCR reaction and cloned in to pRSET construct, that contains a His-tag followed by an E.coli lipoyl-domain at N-terminal to enhance the over expression and for the purification purpose.
8.2 Description of media used
For E.coli cell cultures, 2x TY medium (5g/l NaCl, 10g/l yeast extract and 16g/l tryptone) was used. For preparing 2x TY plates, 15g/l agar was used. Both Plates and media were supplemented with antibiotics ampicillin (100μg/ml, Astra Zeneca, Sweden) and chloramphenicol (34μg/ml, Calbiochem, USA).
8.3 Description of E.coli cells used for cloning and over expression
Chemically competent E.coli XL1 Blue cells (Stratagene, USA) were used for cloning purpose.
For the over expression of proteins, E.coli BL21 cells (DE3) PlysS (Invitrogen, USA) were used.
For the transformation, 2-μl plasmid- DNA was added to 10 μl of competent E.coli BL21 cells thawed on ice. After the addition, the cells were incubated on ice for 30 min and were heat–
shocked at 42°C for 44 seconds; followed by incubation on ice for another 3 min. 150 to 200μl 2x TY medium was added and the cells were incubated at 37 °C for 30 minutes in a rotary shaker. Then, cells were plated on 2x TY plates and incubated at 37 °C overnight.
8.4 Cloning protocol to generate 45 E7 and p21CIP1-C expression constructs
Primers for 45E7-FHPV 45_F ctc gag gga tcc atg cat gga ccc cgg gaa aca ctg HPV 45_R aag ctt gaa ttc tta ttg gtt agt tgc aca cca
Primers for p21CIP1-C
P21_C_F ctc gag gga tcc ggt cga aaa cgg cgg cag acc P21_C_R aag ctt gaa ttc tta ggg ctt cct ctt gga gaa
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Table 2 . PCR reaction ingredients to amplify gene products of 45 E7-F and p21CIP1-C
Template 1µl
Forward primer 0.5µl
Reverse primer 0.5µl
DMSO (dimethyl sulfoxide) 1µl
Pfu buffer (10x, Stratagene) 5µl
dNTP (2.5mM stock 2.5µl
Pfu turbo (Stratagene) 0.5µl
D.H2O Make up to 50µl
.
PCR reaction to amplify 45 E7-f and p21CIP1-C protein
PCR mixture was prepared as given in (Table 2.) and the reaction was set based on the protocol given in the (Table 3). The denaturation temperature was set to 94°C (step 1 and 2) followed by annealing temperature at 64°C (step 3) and elongation 72 °C (step 4 and 5). The repetitive amplification (steps 2 to 4) was set to 35 cycles.
Table 3. PCR protocol
STEPS TEMPERATURE IN °C TIME IN SECONDS
1 95 120
2 94 30
3 64 30
4 72 45
5 72 600
The PRC product was checked on 1% agarose gel in 0.5x TBE buffer (45 mM Tris- borate, 1 mM EDTA (ethylene diamine tetra acetic acid).
Ligation of the amplified gene products were done by adding 6µl DNA -plasmid with 1 µl of PCR gene product and 2µl of ligation enzyme and kept overnight at room temperature. The construct was transformed in to E.coli XL1 cells and plated on 2 X TY plate and left over night at 37°C to get colonies.
The cloned constructs were purified with the Plasmid Mini Kit (Omega Bio-Tek, VWR, Sweden) and sequenced at Uppsala genome centre (Uppsala University)
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8.5 Protein over expression
45 E7-F and p21-C constructs were transformed to E.coli BL21 (DE3) pLysS cells and kept overnight at 37 °C as described in the above scheme. The cells were grown in small culture scale in 10 ml 2xTY medium for 3-4 hours at 37 °C. Then transferred to large-scale culture in 800 ml 2x TY medium for 3-4 hours at 37 °C and grown up to OD 600 nm 0.8-1. Induction was done with 1mM IPTG (isopropyl β-D thio galacto pyranoside) from Saveen Werner AB, Sweden at 18
°C and the cultures were grown overnight for expression. Cells were harvested by centrifugation at 5000 rpm for 20 min at 4 degree C and were resuspended in 50 mM Tris-HCl 400 mM NaCl pH 7.5 buffer containing 2mM 2-mercaptoethanol.
8.6 Protein purification
Harvested E7 and p21 C cells were sonicated on ice for 6 minutes with a Sonics Vibra Cell from chemical instruments AB followed by the centrifugation at 25000 rpm for one hour in a Beckman Avanti J-25 Centrifuge (Beckman Coulter AB). After centrifugation, cell lysates were filtered through 0.45 μm and then 0.2 μm filters (Sarstedt) and subjected to immobilized metal affinity chromatography. The IMAC purification was carried out on a column of Chelating Sepharose Fast Flow (GE health care) with immobilized Ni+2.
8.7 Purification of E7
The filtered E7 cell lysate was loaded on the Ni+2column equilibration with 50mM Tris-HCl, 400 mM, 2 mM mercaptoethanol and 20 mM imidazole pH 7.9. Protein was eluted with 250 mM imidazole pH 7.9 in 8ml fractions after washing the column with 300 ml of the same equilibration buffer. Using Thermo Scientific Nanodrop2000c Spectrophotometer (Saveen Werner) absorbance was measured at 280nm. Peak Fractions containing protein was pooled and dialyzed overnight against 2 liters 50 mM Tris-HCl, 2 mM mercaptoethanol pH 8.5.
The dialysed sample was subjected to Ion-exchange chromatography on HPLC, AKTA explorer (Pharmacia Biotech) system. The anion exchange Q column (GE Health care) equilibrated with 50 mM Tris-HCl, 2mM mercaptoethanol pH 8.5 was used to purify E7. Gradient elution was carried out at 0-500 mM NaCl, 50mM Tris-HCl, 2 mM mercaptoethanol pH 8.5 buffer concentrations and the protein was eluted at 80% of NaCl.
The protein was not pure after this step; therefore the sample was subjected to reversed phase chromatography using a C8 column (GE Health care) equilibrated with 0.1% TFA on HPLC, AKTA explorer (Pharmacia Biotech) system. Gradient elution was carried out at 0-100% in 0.1% TFA in water and the protein was eluted at 20% of TFA. The purity was checked using SDS-PAGE. But the protein was not pure enough to carry out the biophysical experiments;
therefore the sample was lyophilized and dissolved in ion-exchange buffer and again loaded on to anion exchange Q column (GE Health care) equilibrated with 50 mM Tris-HCl, 2 mM
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mercaptoethanol pH 8.5 to get highly pure E7. Gradient elution was carried out at 0-500 mM NaCl, 50 mM Tris-HCl, 2mM mercaptoethanol pH 8.5 buffer concentrations and the protein was eluted at 80% NaCl. The purity of E7 purification was checked on the SDS-PAGE. Gels were stained with coomassie brilliant blue.
8.8 Purification of p21CIP1-C
Filtered p21-C cell lysate was loaded on the Ni+2 column equilibrated with 50 mM Tris-HCl, 400 mM NaCl, 2 mM mercaptoethanol and 20 mM imidazole pH 7.9. The anion exchange Q column (GE Health care) equilibrated with 50 mM Tris-HCl, 2 mM mercaptoethanol pH 8.5 was used to purify E7. Then, sample was subjected to lipo-tag digestion with thrombin enzyme for 4 hours at 37°C. The cleavage was checked on SDS gel after digestion. Then, IMAC purification was carried out to purify p21-C from the digested lipo-domain. At this point, the peptide was not completely pure therefore; reversed phase column equilibrated with 0.1% TFA on HPLC, ÄKTA explorer (Pharmacia Biotech) system was used to get highly pure p21-C. Pure peptide was eluted at 50% concentration TFA buffer. The purity was checked by SDS-PAGE. Gels were stained with coomassie brilliant blue. Columns selected for ion-exchange chromatography and pH of the buffers to purify these proteins were based on the theoretical pI of the proteins generated by Expasy Proteomics tool ProtParam (http://www.expasy.ch/cgi-bin/protparam).
SDS-PAGE
Protein samples for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were prepared with 4x Laemmli loading dye and 15-20 μl sample was loaded onto the SDS- PAGE. Solutions for stacking and separating gels were prepared according to the following Scheme (Table 4.)
Table 4. SDS gels preparation
Ingredients Separating gel Stacking gel Acryl amide solution (30% acryl
amide/0.8% bisacryl amide [AppliChem, Germany])
2500µl 375 µl
80% (v/v) glycerol 625 µl
0.5 M Tris-HCl pH 6.8, 0.4%
(w/v) SDS
1250 µl 1.5 M Tris-HCl pH 8.8, 0.4%
(w/v) SDS
625 µl APS (ammonium peroxide sulfate;
40%
(w/v)
15 µl 4 µl
TEMED ( N, N, N’, N’- Tetramethylethan-
1,2-diamin;
Sigma, Germany)
15 µl 4 µl
H2O 595 µl 1500 µl
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After SDS-PAGE run, gels were kept for coomassie staining in (0.1% (w/v) Coomassie [VWR, Sweden], 10% (v/v) acetic acid [Merck, Germany], 40% (v/v) ethanol [Solveco, Sweden]) staining solution. Gels were stained overnight and were rinsed with water and destained in (10%
acetic acid and 10% Isopropanol) destaining solution. The protein bands appeared 25 to 50 minutes after destaining.
8.9 Stability experiments
Stability of E7 and p21CIP1-C was measured by equilibrium denaturation experiments. In this method, fluorescent measurements were recorded at equilibrium in both native and denatured states of protein. All experiments were carried out in potassium phosphate buffer pH 7.5.
Experimental buffers such as (A) 8.5 M urea in 50mM potassium phosphate pH 7.5 and (B) 50 mM potassium phosphate pH 7.5 was prepared. The final urea concentration in buffer (A) and the highest concentration used for the experiment were measured using a refractometer. 5μM protein was prepared in 10 ml of buffer (A) and 10 ml of buffer (B). Protein was gradually denatured by step-wise increase in the concentration of urea buffer (A). Before taking each measurement, equilibrium condition was obtained by mixing the solution for 2 minutes.
Tryptophan was excited at 280 nm and the emission was recorded from 300 to 400nm wavelength on an SLM 4800 spectrofluorimeter (SLM instruments, IL). Fluorescence intensity at 345nm was then plotted against urea concentration fitted into the following equation of solvent denaturation of a two-state protein (Fersht 1999) in KaleidaGraph4.0.
F = (αN + βN [denaturant] + (αD + βD [denaturant]) exp [mD-N ([denaturant] – [D] 50%)/RT]/
(1+ exp [mD-N ([denaturant] – [D] 50%) /RT]). equation.1 Where,
F - Observed spectroscopic signal, αN - intercept of the native state baseline
αD - are intercept of the denatured state baselines, βN and βD - slopes of the respective baselines,
mD-N - free energy of unfolding on denaturant concentration
[D] 50% - midpoint (concentration of urea at which 50% of the protein is denatured).
The fitting errors were given for the individual experimental measurements of mD-N and [D]
50%. The value of m reflects the cooperatively of the transition and the exposure of the denatured state relative to the native state (33). ΔGD-N [urea] 50%
in H2O was calculated with free- fitted mD-N values showing the dependence of free energy on denaturant concentration when the protein is 50% denatured.
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9. ACKNOWLEDGEMENTS
First of all, I thank Prof. Per Jemth for giving me this valuable opportunity to work in his laboratory. His consistent encouragement and proper guidance throughout the project helped me to learn the fundamentals of protein science. I would like to thank personally Dr. Celestine Chi for teaching me everything right from the scratch and made me to understand the goals and outcomes of the project.
Secondly, I thank all my lab members Jakob Dogan, Raza Haq, Greta Hultqvist, Andreas Karlsson, for their support and assistances all through this project.
The working environment was really fantastic and I enjoyed it!
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