3 Material and Methods
3.2 Methods
3.2.3 Recombinant Protein Overexpression and Purification
The use of insect cell culture (High Five, Sf9) offers a good compromise between high expression yields, proper protein folding and most eukaryotic PTMs for recombinant protein overexpression. Here, two different insect cell lines were used. Sf9 cells show lower expression levels than the other used insect cell line, High Five. Thus, baculovirus recombination and virus amplification was performed with Sf9 cells. While the high recombinant protein expression yield of High Five cells was used for the overexpression of XPA, but not PARP-1, which showed already in Sf9 cells a very high expression yield.
3.2.3.2 Generation of Overexpression Constructs
In the course of this thesis it was aimed at the overexpression and purification of five fragments of XPA, F1 (aa 1-97), F2 (aa 60-140), F3 (aa 98-211), F4 (aa 180-247) and F5 (aa 226-273). Therefore, constructs for baculovirus recombination had to be cloned first. Using pSL1180::His-XPA as a template, the DNA coding for the fragments were amplified by PCR (Table 3.2). The primers used for the PCR were 5’ elongated to introduce NotI and BmtI restriction sites at both ends of the amplicon.
After PCR amplification, the DNA was restriction digested, agarose gel purified and ligated into the pVL1392::His baculovirus recombination vector, which was as well digested with NotI and BmtI (for scheme see Figure 4.2A).
Table 3.2: Thermocycling parameters for XPA-fragment amplification.
Step Temperature Time
1. Initial denaturation 95°C 120 s
2. Denaturation 95°C 20 s
3. Initial primer annealing X 30 s
4. Elongation 70°C 30 s
5. Denaturation 95°C 20 s
6. Specific primer annealing Y 30 s
7. Elongation 70°C 30 s
8. Final elongation 70°C 10 min
9. Storage 8°C ∞
Steps 2-4 were repeated 10 times, steps 5-7 were repeated 18 times. X represents the initial annealing temperature, Y the specific annealing temperature of each fragment (see Table 3.3).
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Table 3.3: Initial (X) and specific (Y) annealing temperatures for each XPA-fragment.
XPA-fragment Initial annealing temperature Specific annealing temperature
Fragment 1 56°C 69°C
Fragment 2 46°C 63.6°C
Fragment 3 41.3°C 63.3°C
Fragment 4 46°C 63.6°C
Fragment 5 51.5°C 65.9°C
3.2.3.3 Baculovirus Recombination
The generation of a baculovirus carrying the information of the to be overexpressed protein was performed in Sf9 cells. The BD BaculoGold™ Baculovirus Expression system was used for transfection of the recombinant vector and the linearized baculovirus DNA. The subsequent recombination of these enabled the generation of a recombinant baculovirus budding off of the transfected cells.
Therefore, 1x106 Sf9 cells were seeded in a 35 mm dish. After 4 h, cells were attached and the medium was aspirated. 380 µl Transfection buffer A (TB-A) was added to the cells, evenly distributed and incubated at RT. Meanwhile, in a 1.5 ml reaction tube 2 µl linearized baculovirus DNA and 1.2 µg recombinant plasmid (e.g. pVL1393::His-XPA) were mixed and incubated at RT for 5 min. Thereafter, 380 µl Transfection buffer B (TB-B) was added and carefully mixed. The DNA/TB-B mixture was then added dropwise to the cells with gentle rocking of the plate every 3-4 drops to mix it with TB-A. Sf9 cells were incubated for 4 h at 27°C before removing the transfection mix and adding 1.2 ml TC-100 (FCS; gentamycin). Cells were then incubated again at 27°C and regularly checked for signs of infection (enlarged, circular cells). After 4-8 days, but before massive cell lysis was observed, supernatant was taken and centrifuged (5 min at 126 xg). Supernatant was stored in the dark at 4°C until usage or flash frozen in liquid nitrogen and kept at -80°C for long term storage.
3.2.3.4 Baculovirus Titer Amplification
The generation of baculovirus gave rise to around 1 ml supernatant with a low viral titer. For efficient protein expression this titer had to be amplified in 3-4 rounds, optimally resulting in a viral titer of
≥1x108 pfu/ml. Repetitive infection cycles, as well as infections with high MOIs (multiplicity of infection), increase the portion of deletion mutant viruses, no longer carrying the information for the recombinant protein. Thus, not more than 5 serial amplifications were performed and in each amplification cycle it was taken care not to use a too high MOI for initial infection (MOI ≤ 1, if MOI was known).
For amplification of the viral titer, 2x107 cells were seeded in 100 mm dishes and allowed to settle for 4 hours. Thereafter, the medium was removed and 100-250 µl virus supernatant was added and filled up with insect growth medium to 3 ml. Dishes were incubated at 27°C and rocked gently back and forth every 10 min. After 1 hour 7 ml insect growth medium was added and the cells were cultured for 4-6 days and regularly checked for signs of infection. The virus containing supernatant was taken off and centrifuged for 5 min at 126 xg to remove cell debris. Supernatant was then aliquoted and stored as described above or used for another round of amplification.
3.2.3.5 Baculovirus Titer Determination
After the third titer amplification cycle the viral titer was determined. This was done by identification of infected cells in a dilution series via immunofluorescence based microscopy.
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3x104 Sf9 cells/well were seeded in a 96-well plate and incubated for 2 hours at 27°C. After the cells adhered, medium was replaced by a dilution series of virus containing supernatant (100 – 10-7) and the negative control (only medium). After three days medium was removed and cells were carefully washed with PBS. Cells were fixed in 4 % PFA for 30 min, then it was removed and residual PFA was inactivated with 100 mM glycine (in PBS) for 5 min. Glycine was replaced by a permeabilization buffer (0.5 % (v/v) TritonX-100 in PBS) in which cells were incubated again for 5 min. Cells were washed twice with PBS, and blocked for 1 hour in PBS-MT (5 % (w/v) skim milk; 0.05 % (v/v) Tween20 in PBS) at RT. Incubation in first antibody (1:300 for PARP-1 (CII10), 1:300 for XPA (FL-273), 1:200 for XPA-Fragments with an anti-His-Tag antibody (His.H8) was done at 37°C for 1 h and afterwards washed thrice for 5 min in PBS. Cells were then incubated overnight at 4°C in secondary antibody (1:400 goat-α-mouse Alexa Fluor 488 or 546 for CII10 and His.H8, 1:400 goat anti-rabbit Alexa Fluor 488 or 546 for FL-273). After three PBS washing steps, 5 min each, nucleus was stained with Hoechst33342 (200 ng/ml in PBS) at RT for 10 min. Cells were washed again thrice in PBS and stored at 4°C in the dark until microscopic analysis.
The likelihood of a virus infection was determined by setting up the ratio of infected to uninfected cells per well (P(n≥1)). Only virus dilutions with ratios between 0.1 and 0.9 were considered trustworthy since high MOIs can lead to cell lysis and thus mislead in the calculation of viral titer.
The following Poisson distribution describes the likelihood (P(n)) that a given number of virus particles (n) and virus to cell ratio (m) will infect a cell:
= ∗
!
When no virus is present, the number of uninfected cells (P(n=0)) can be described as
= 0 =
which can be solved for the MOI (m):
= −ln [ = 0 ]
Further, the number of uninfected cells can be calculated from the likelihood of cells becoming infected by one or more virus particles:
= 0 = 1 − ≥ 1
Using these equations, the titer of the virus containing supernatant can be calculated:
= ∗
∗ =− [ = ] ∗
∗ =− [ − ≥ ] ∗
∗
T=Virus titer )*+,-.
=Multiplicity of infection MOI
c=number of seeded Sf9 cells per well cfu
- 51 - D=Dilution of virus supernatant
V=Volume of the well during infection
3.2.3.6 Test Expression of Overexpressed Recombinant Proteins
As soon as titer determination resulted in more than 1x108 pfu/ml, the virus supernatant was next used to determine the MOI and expression period at which recombinant protein expression was optimal.
Therefore, 3x105 High Five or Sf9 cells were seeded in 35 mm petri dishes. On the next day, the medium was aspired and the cells were inoculated in baculovirus with MOIs I-III. 24, 48 and 72 h after infection, cell lysates were prepared. Cells were detached with a cell scraper, centrifuged (3 min; 126 xg) and the resulting pellet was resolved in 500 µl hot 2x SDS LD. After a 5 min incubation step at 95°C, 15 µl per sample was subject to SDS-PAGE (chapter 3.2.4.2). The proteins were blotted either semidry or wet and the membrane was incubated for 1 h in 5 % skim milk (in TBS-T). Detection of overexpressed protein was performed by incubation for 1 h at RT in specific 1st antibody (Fragments: His.H8, 1:1,000;
XPA: Fl-273, 1:1,000; PARP-1: CII10, 1:300 in blocking buffer). The blot was washed thrice for 5 min in TBS-T. Staining with 2nd antibody was conducted for 1 h at RT (GαM- or GαR-HRP, 1:2,000 in blocking buffer). Again, the blot was washed thrice in TBS-T for 5 min before being incubated in ECL Advance and ECL (1:10). Chemiluminescence was detected with an ImageQuant LAS 4000 mini.
3.2.3.7 Overexpression of Recombinant Proteins
Overexpression of recombinant PARP-1 protein was performed in Sf9 cells. Therefore, a defined number of Sf9 cells were seeded the day before inoculation. Baculovirus was added to the cells at an MOI of I and cells were cultivated for three days. Thereafter, cells were carefully washed with cooled PBS, detached with a cell scraper and centrifuged (5 min; 126 xg). The resulting pellet was flash frozen and stored at -80°C until further usage.
Overexpression of His-XPA and His-XPA-Fragments was conducted in the same manner with small modifications. First, overexpression was performed in High Five cells. Second, a MOI of III was identified to yield the highest expression levels.
3.2.3.8 Purification of Recombinant PARP-1
Most recombinant proteins tend to be temperature sensitive, change or lose their protein function and proper folding and degrade faster at higher temperatures. Thus, all purifications described below were performed when possible in a cool room at 8°C and otherwise on ice. All used buffers were cooled to 8°C and due to the temperature-dependent pH of some buffers, pH was only adjusted when already cooled to working temperature. Because of their instability in aqueous solutions, some chemicals, like the protease inhibitors cOmplete, EDTA free or PMSF, or the reducing agents 2-mercaptoethanol and DTT, were only added directly before usage. Further, all buffers were sterile filtered before usage.
Purification of proteins was performed as fast as possible and at a time. Repeated freeze-thaw cycles as well as stretched out periods on ice were avoided when possible.
PARP-1 was expressed from a pVL1393 vector and no epitope tag was grafted to the recombinant protein. Thus, strategies to purify PARP-1 included its specific solubility as well as its DNA-binding ability. The purification procedure for PARP-1 can be divided in the functional steps following cell lysis: DNA and protein precipitation, sephadex gel filtration chromatography, DNA cellulose affinity chromatography and dialysis.
- 52 - Cell Lysis:
Sf9 cell pellets were put in ice just until before thawing. PMSF and 2-mercaptoethanol were added to PARP-1 purification buffer 1 (25 mM Tris-HCl, pH 8.0; 50 mM glucose; 10 mM EDTA; 1 mM PMSF;
1 mM 2-mercaptoethanol) and the cells were finally thawed and resuspended in 1 ml buffer per 1.5x107 Sf9 cells. Tween20, NP-40 and NaCl were added to final concentrations of 0.2 %, 0.2 % and 0.5 M, respectively. The cell lysate was mixed thoroughly and incubated on a tube rotator for 20 min at 4°C.
Thereafter, to remove the cellular debris the lysate was ultracentrifuged (20,000 xg; 20 min; 4°C) and the supernatant was taken off for further purification.
DNA and Protein Precipitation:
To precipitate the DNA, 1 mg protamine sulfate per ml lysate was added to the supernatant and the mixture was centrifuged at 20,000 xg (10 min; 4°C). Again, the pellet was discarded and a two-step protein precipitation was performed. In a first step the lysate was 30 % saturated with ammonium sulfate (155 mg per ml cell lysate) to salt out less soluble proteins. The lysate was centrifuged (25,000 xg; 20 min; 4°C) and the pellet was discarded. The precipitation of PARP-1 was achieved by adding 259 mg ammonium sulfate per ml supernatant (80 % saturation). The mixture was again centrifuged (20,000 xg; 15 min; 4°C) and the supernatant was discarded.
Sephadex Gel Filtration Chromatography:
The protein pellet was solved (1 ml per 4x107 cells) in PARP-1 purification buffer 2 with freshly added PMSF and 2-mercaptoethanol (100 mM Tris-HCl, pH 7.4; 10 % glycerol; 0.5 mM EDTA; 1 mM PMSF;
2 mM 2-mercaptoethanol).
DTT and 2-mercaptoethanol were added to PARP-1 purification buffer 3 (50 mM Tris-HCl, pH 8.0;
200 mM KCl; 1 mM EDTA; 1 mM DTT; 10 mM 2-mercaptoethanol). 1 g of Sephadex G-100 superfine material was resuspended in 40 ml PARP-1 purification buffer 3 and incubated for 30 min on a tube rotator at RT. The sephadex medium was put in the Poly-prep column and the liquid was allowed to drain. Drying out of the medium was avoided. Next, the resuspended protein pellet was applied to the column and the flow-through was collected. 2x 15 ml of PARP-1 purification buffer 4 with freshly added 2-mercaptoethanol and PMSF (50 mM Tris-HCl, pH 8.0; 5 mM MgCl2; 0.5 mM EDTA; 5 % glycerol; 1 mM PMSF; 12 mM 2-mercaptoethanol) were applied to the column. 2 ml elution fractions were collected and tested for PARP-1 by SDS-PAGE (10 %) and subsequent Coomassie staining and western blot. All PARP-1 containing fractions were pooled for the next purification step.
dsDNA Cellulose Affinity Chromatography:
0.3 g dsDNA cellulose was resuspended in 5 ml PARP-1 purification buffer 4 (plus 100 mM KCl, 1 mM PMSF and 12 mM 2-mercaptoethanol) and incubated on a rotator for 30 min at RT. The suspension was centrifuged (1,000 xg; 5 min; RT) and the supernatant discarded. The material was washed again in 7 ml PARP-1 purification buffer 4 (as above), incubated for 30 min on a rotator and centrifuged (1000 xg; 5 min; RT). The supernatant was removed, the medium was resuspended in 5 ml PARP-1 purification buffer 4 (as above) and the solution was put into a Poly-prep column and allowed to settle down. After the liquid was drained out (again drying out of the chromatography medium was avoided) the pooled PARP-1 fractions of the gel filtration were applied to column and the flow-through was collected. The column was washed with 6 ml PARP-1 purification buffer 4 (with PMSF and 2-mecaptoethanol as above) and the flow-through was collected again. Another washing step with 3 ml PARP-1 purification buffer 4 (plus 100 mM KCl, 1 mM PMSF and 12 mM 2-mercaptoethanol) was
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performed and the eluate was collected, this was repeated once. Elution of PARP-1 started when 3 ml PARP-1 purification buffer 4 (plus 200 mM KCl, 1 mM PMSF and 12 mM 2-mercaptoethanol) was applied to the column and the flow through was collected. 2 ml PARP-1 purification buffer 4 (plus 400 mM KCl, 1 mM PMSF and 12 mM 2-mercaptoethanol) was added and fractions were collected. A final PARP-1 elution step with 2 ml PARP-1 purification buffer 4 (plus 1000 mM KCl, 1 mM PMSF and 12 mM 2-mercaptoethanol) was performed and again the eluate was collected. As for the gel filtration fractions dsDNA affinity fractions were analyzed by SDS-PAGE (10 %) and subsequent Coomassie staining and western blot.
Dialysis and Protein Storage:
PARP-1 containing fractions were supplemented with glycerol to a final concentration of 20 % and separately dialyzed (MWCO 12-14 kDa) in 2 liter PARP-1 storage buffer (20 % glycerol in PBS) on a magnet stirrer at 4°C. After the first hour the buffer was renewed and dialysis continued overnight.
Before the protein was aliquoted, flash frozen and stored at -80°C, protein concentration was determined via BCA assay.
3.2.3.9 Purification of Recombinant His-XPA
Recombinant XPA was expressed with an N-terminal hexahistidine-tag. For the first purification step, the affinity of this his6-tag to immobilized divalent metal ions (Ni2+) was exploited. In a second step, XPA’s DNA-binding ability was utilized and the protein was further purified using its affinity to heparin.
For the purification of recombinant full-length His-XPA the methodology switched from gravity flow chromatography to FPLC (Fast Protein Liquid Chromatography) based protein purification. The therefore newly established protocol involved the steps of cell lysis, crude extract clearing, two steps of affinity chromatography (Ni sepharose and heparin) and a final step of protein concentration and buffer exchange.
Cell Lysis:
10 ml ice cold XPA lysis buffer (50 mM KH2PO4; 100 mM KCl; 0.5 % NP-40; 10 % glycerol; 5 mM imidazole; 0.5 mM PMSF; 1x cOmplete, EDTA free; pH 8.0) per 1 g High Five cells was added to the still frozen cell pellet. PMSF and 1x cOmplete were added and the pellet was thawed and dissolved on ice. The cell suspension was incubated for 20 min at 4°C on a tube rotor. Cellular DNA was fragmented by sonication. Therefore 50 ml centrifugation tubes were filled with about 20 ml cell lysate and were sonicated 8x 30 s, divided by 30 s breaks, with a sonication needle in an EtOH-ice water bath (30 % duty cycle, maximum output).To clear the crude extract, the cell lysate was centrifuged for 30 min at 17,400 rpm (37,000 xg in a SW32Ti rotor at an average distance) at 4°C. The supernatant was further filtered through a 0.45 µm syringe filter and diluted 1:4 in XPA lysis buffer.
General Preparation of ÄKTA FPLC:
Before the cell lysate was loaded onto the superloop, the FPLC ÄKTA had to be prepared. All four pumps, the superloop and all flow paths were washed thoroughly with MilliQ water at a flow rate of 1 ml/min to remove all remaining ethanol. A 1 or 5 ml HisTrap FF column was connected to the FPLC drop-to-drop and the column was washed with at least 5 CV MilliQ water. Finally, the two Buffer-A pumps, the superloop and all flow paths, including the column, were washed with XPA purification
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buffer A (50 mM KH2PO4; 100 mM KCl; 10 % glycerol; pH 8.0); the two Buffer-B pumps with XPA purification buffer B (as XPA purification buffer A plus 500 mM imidazole).
HisTrapFF Affinity Purification (IMAC):
The superloop was loaded with the diluted cell lysate and a standardized protocol was started. With a flow-rate of 0.5 ml/min the lysate was run over the HisTrap FF column (5 mM imidazole). His-tagged XPA binds to the material, while most other proteins flow through. His-XPA was eluted at 200 mM imidazole (15 CV) and thereafter the column was flushed with 250 and 500 mM imidazole to remove all remaining proteins. Flow-through was collected in 50 ml fractions, while all washing and elution steps were collected in 1 ml fractions. Fractions were tested for His-XPA by running a SDS-PAGE (12
%) and subsequent Coomassie staining.
The ÄKTA FPLC, all pumps and flow paths, as well as the column was washed thoroughly with MilliQ and subsequently with 70 % EtOH.
Table 3.4: Parameters of HisTrap FF affinity chromatography (His-XPA).
Buffer A: 0 mM imidazole; Buffer B: 500 mM imidazole.
HisTrap FF
The FPLC was prepared as described for HisTrap FF affinity purification, but with XPA purification buffer C & D (25 mM Tris-HCl; 100 mM KCl; 10 % glycerol; 1 mM DTT; 0.5 mM PMSF; pH 8.0;
plus 1 M NaCl for buffer D) and a HiTrap Heparin HP column. XPA containing fractions were pooled, diluted 1:2 in XPA purification buffer C and loaded onto the superloop. Again, a standardized protocol was run. At a flow rate of 0.5 ml/min the pooled HisTrap FF eluate was loaded on the HiTrap Heparin HP column (40 mM NaCl). Flow-through was collected in 50 ml, all following washing and elution steps in 1 ml fractions. The column was washed with 15 CV XPA purification buffer (85 % buffer C &
5 % buffer D; 150 mM NaCl) and with 10 CV XPA purification buffer (80 % buffer C & 20 % buffer D, 200 mM NaCl). XPA was eluted with 400 mM NaCl (10 CV). Finally, the column was washed with 1 M NaCl to remove all remaining proteins. Fractions were tested for His-XPA by SDS-PAGE (12 %) and Coomassie staining.
The ÄKTA FPLC, all pumps and flow paths, as well as the column was washed thoroughly with MilliQ and subsequently with 70 % EtOH.
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Table 3.5: Parameters of HiTrap Heparin HP affinity chromatography (His-XPA).
Buffer C: 0 mM NaCl; Buffer D: 1 M NaCl.
His-XPA containing fractions were either concentrated using Vivaspin 6 concentrators (3 kDa MWCO) or separately dialyzed (Slide-A-Lyzer Dialysis Cassette, 3 kDa MWCO) in 2 liter XPA storage buffer (25 mM Tris-HCl, pH 7.4; 10 % glycerol) on a magnet stirrer at 4°C. After the first hour the buffer was renewed and dialysis continued overnight. Before the protein was aliquoted, flash frozen and stored at -80°C, protein concentration was determined via BCA assay.
3.2.3.10 Purification of Recombinant His-XPA Fragments
The fragments of XPA were expressed with an N-terminal hexahistidine-tag, therefore the first purification step was performed with a HisTrap FF column according to the first purification of full-length His-XPA. Due to the absence of a functional DNA-binding site all fragments of XPA were further purified using size exclusion chromatography (besides XPA-F3, which reached a sufficient degree of purity in one step).
Cell lysis and ÄKTA FPLC preparation were as described for full-length His-XPA. The clarified lysate was then loaded at a flow rate of 0.5 ml/min on the HisTrap FF column (5 mM imidazole). For XPA-F1, the column was then washed with 10 mM imidazole. Three elution steps with increasing concentrations of imidazole (150 mM, 200 mM and 500 mM) followed (Table 3.6). Flow-through was collected in 50 ml fractions, while all washing and elution steps were collected in 1 ml fractions.
Fractions were tested for XPA-F1 by running a SDS-PAGE (12 %) and subsequent Coomassie staining.
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Table 3.6: Parameters of HisTrap FF affinity chromatography (His-XPA-F1).
Buffer A: 0 mM imidazole; Buffer B: 500 mM imidazole.
Buffer A: 0 mM imidazole; Buffer B: 500 mM imidazole.