Protein Expression

4.7 1 Protein expression in E. coli

RPA heterotrimeric complex was expressed and purified from Rosetta cells (Table 6), as described previously²⁹¹. TFIIS and inactive TFIIS:D282AE283A mutant were expressed as follows. After the transformation of BL21 (DE3) RIL (Table 6) and selection on LB agar plates, a colony was used for the inoculation of 500 ml LB medium (Table 4) supplemented with kanamycin (30 µg/ml) and chloramphenicol (34 µg/ml). The culture was grown overnight at 37 °C while shaking. The next day 6L of 2x YF (Table 4) medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (34 µg/ml) was inoculated with 180 ml of the overnight culture and grown at 37 °C until the OD600 of 0.4 – 0.5 was reached. Next, the temperature was lowered to 18 °C and the cells were induced with IPTG (0.5 mM final).

The cells were shaken at 18 °C overnight. Cells were harvested by centrifugation (7000g at 4°C for 20 min) and the supernatant was discarded. Cells were resuspended in the lysis buffer (400 mM NaCl, 20 mM Tris-HCl pH 7.9, 10 % glycerol (v/v), 1 mM DTT, 30 mM imidazole pH 8.0, 0.284 μg/ml leupeptin, 1.37 μg/ml pepstatin A, 0.17 mg/ml PMSF and 0.33 mg/ml benzamidine), flash-frozen in liquid nitrogen and stored at -80 °C.

4.7.2 Protein expression in insect cells

Protein expression in insect cells included several steps after the genes of interest were cloned into the respective 438 vectors: transformation of electrocompetent DH10EMBacY cells, isolation of the bacmid DNA, production of V0 and V1 viruses and protein expression in Hi5 cells, as previously described^292,293.

4.7.3 Transformation of electrocompetent DH10EMBacY cells

100 µl of electrocompetent DH10EMBacY cells were thawed on ice and gently mixed with the 500 ng of DNA dissolved in ddH2O. Cells were incubated with the DNA for 15 min on ice and then transferred to a pre-chilled BIORAD Gene®/MicroTM Pulser cuvette (0.1 cm width). Electroporation was performed with a single pulse (25µF, 1.8 kV). Cells were immediately supplemented with 900 µl of LB medium (Table 4) at RT and transferred to a 15 ml culture tube. Cells were incubated at 37 °C for 5 h while shaking. After the incubation, 50 µl of transformed cells was plated on the LB agar supplemented with gentamycin (10 µg/ml), X-Gal (150 µg/ml) and IPTG (1mM) for blue-white selection. If the integration of the desired DNA into the bacmid DNA was successful, the colonies would appear white, and if the integration was unsuccessful the colonies would turn blue. The plates were incubated for 36 h at 37 °C for a reliable blue-white selection. Single white colonies were

picked and restreaked on the LB agar supplemented with gentamycin (10 µg/ml), X-Gal (150 µg/ml) and IPTG (1mM). If the colonies were still white after incubation at 37 °C for 36 h, we preceded with the isolation of bacmid DNA.

4.7.4 Isolation of bacmid DNA

White colonies were used to inoculate 5 ml of LB medium (Table 4) supplemented with gentamycin (10 µg/ml). The bacterial culture was grown overnight at 37 °C while shaking.

After the incubation, the cells were harvested by centrifugation (3000g at 4 °C for 10 min).

The bacterial pellet was resuspended in 250 µl of P1 buffer (QIAprep Spin Miniprep kit, Quiagen), followed by the addition of 250 µl of P2 buffer (QIAprep Spin Miniprep kit, Quiagen) and incubation at RT for 5 min. After the incubation, 350 µl of N3 buffer (QIAprep Spin Miniprep kit, Quiagen) was added and the mixture was centrifuged at 21 000g and 4

°C for 10 min. The supernatant was transferred to a fresh 1.5 ml tube and centrifuged for another 10 min to remove the remaining precipitates. The supernatant was supplemented with 700 µl of cold isopropanol and gently mixed. The bacmid DNA was precipitated at -20

°C overnight. Next day, the DNA was pelleted by centrifugation at 21 000g and 4 °C for 15 min. The pellet was washed with 500 µl of cold 70% ethanol. After the ethanol removal, the pellets were covered with 30 µl of fresh 70% ethanol and stored at -20 °C until the transfection of insect cells.

4.7.5 Transfection of Sf9 cells with bacmid DNA and V0 virus production

All consecutive steps were performed under a sterile hood (Biowizard Golden Line, Kojair) because the insect cell cultures are extremely susceptible to fungal infections. The precipitated bacmid DNA was transferred into a sterile hood and the ethanol was removed.

The DNA pellets were left to dry for 10-15 min followed by the addition of 20 µl of water without resuspending the DNA to prevent DNA fragmentation. For transfection, the X-treme GENE^TM 9 transfection agent (10 µl) was mixed with Sf-900TM III SFM medium (100 µl).

The transfection agent solution was added to the dissolved bacmid DNA and the solution was further diluted with 200 µl of the Sf-900TM III SFM medium. The solution was incubated at RT for 1h. In the meantime, the Sf9 cells were prepared for the transfection.

Density of the Sf9 cell culture and the cell viability was determined with the CASY® Modell TT Cell Counter and Analyzer System equipped with a 150 mm capillary (OMNI Life Science) according to manufacturer`s instructions. The cell density was adjusted to 1.0x10⁶ cells/ml and 3 ml of cells was added to each of the 6 wells in a 6-well plate (Greiner). Cells were incubated at 27 °C until the bacmid DNA was ready for transfection. The 300 µl of the bacmid solution was then added to 2 wells containing Sf9 cells in a dropwise manner (150

particular DNA construct. As a control, one well contained the non-transfected cells and one well contained only the medium. Transfected cells were incubated at 27 °C in the dark without agitation. Efficiency of transfection was monitored with a fluorescence microscope (Leica). Since the bacmid DNA also contains the EYFP gene driven by the same viral promotor that drives the expression of the gene of interest, increase in fluorescence was indicative of a successful transfection. Usually, the cells were incubated for 2-3 days before the fluorescence increased. If a reasonable number of fluorescent cells was observed (at least 50 cells per well) before the fourth day, the supernatant containing the V0 virus was removed in the sterile hood, transferred to a 10 ml tube and stored at 4 °C in the dark.

4.7.6 V1 virus production

V0 virus is still too weak to be used for a protein expression, so the virus is further propagated to produce the stronger V1 virus. 25 ml of the Sf9 or Sf21 insect cell culture (Table 7) at a density of 1.0x10⁶ cells/ml was infected with 0.5-3 ml of the corresponding V0 virus. After the infection, the density, the cell viability and the YFP fluorescence was monitored daily.

The first day at which the cells do not divide is considered the day of proliferative arrest (DPA). Ideally, the cells divided once after the infection with the V0 virus, followed by DPA.

However, if the initial virus is weak, the DPA is reached only after several days. In the meantime, cells were kept at the density of 1.0x10⁶ cells/ml. When DPA was reached, cells were usually grown for additional 48-72 h which was accompanied by an increase in cell diameter (from 25 to 28 µm for Sf21 cells and from 18 to 20 µm for Sf9 cells), a decline in cell viability and an increase in YFP fluorescence. The level of YFP was monitored with an Infinite® M1000 Pro micro-plate reader (Tecan) using excitation and emission wavelengths of 514 nm and 527 nm, respectively. When the viability dropped below 90 % (usually around 80 – 85 %), the cells were harvested by centrifugation (320g, 15 min, 4 °C). The supernatant (V1 virus) was decanted in the hood into a 50 ml falcon tube and stored at 4 °C in the dark.

4.7.7 Protein expression (V2 virus production)

V1 virus was used for the large-scale protein expression. Here we used primarily Hi5 cells for protein expression as Hi5 cells tend to produce larger amounts of protein. 600 ml of Hi5 cell culture at the cell density of 1.0 x 10⁶ cells/ml was infected with 0.3 – 2 ml of V1 virus, depending of the strength and the age of the virus. Usually the amount of the V1 virus added to Hi5 cells was optimized so that the cells divided exactly once before the DPA. Here, we used only one virus for infecting Hi5 cells, except for the expression of the core TFIIH when two viruses were used, one encoding subunits XPB, p62, p52, p44, p34 and TTDA, and one encoding XPD. The cell density, the cell viability and the YFP levels of infected cells were monitored daily. The cell density was kept at 1.0 x 10⁶ cells/ml throughout the expression

period. After the DPA was reached, cells were propagated for additional 48 – 72 h. When the cell viability dropped between 80 and 85 %, cells were harvested by centrifugation (238g, 20 min, 4 °C), resuspended in lysis buffer, flash frozen in liquid nitrogen and stored at -80

°C.