5. MATERIAL AND METHODS
5.1. Chemicals and Buffers
UbcX dissociates XErp1 from the APC/C Material and Methods
55
5. MATERIAL AND METHODS
5.1. Chemicals and Buffers
All materials and reagents were obtained from commercial suppliers and used without further purification, unless specified. Chemicals were at least of purity grade p.a. (pro analysi). Buffers and solutions were prepared with deionized water from a Milli-‐Q system (Millipore) and either autoclaved or sterile filtered before use. Standard buffers utilized in this study are reported below.
Phosphate Buffered Saline (PBS): 137 mM NaCl2, 2.7 mM KCl2, 10.2 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4
Tris Buffered Saline (TBS): 10 mM Tris-‐HCl pH 7.6, 150 mM NaCl2
3x laemmli buffer (3xlb): 180 mM Tris HCl pH 6.8, 30% (w/v) Glycerole ,10 % (w/v) SDS
5.2. Plasmids
5.2.1. Plasmids generated in this study
The plasmids that have been used in this for protein expression or coupled in vitro transcription/translation are listed below referring to the TUM number in the Mayer laboratory plasmid collection:
Table 5.1.: Plasmids
Name Insert Specification Vector Tag Reference
TUM606 hu_securin wt,fl pCS2-‐F/A -‐ N. Rauh
TUM1144 hu_UbcH10 wt,fl pQE80-‐F/A 6xhis E. Hörmanseder
TUM1145 hu_UbcH10 C114S, fl pQE80-‐F/A 6xhis E. Hörmanseder
TUM1165 Xe_Usp44 wt, fl pQE80-‐F/A 6xhis E. Hörmanseder
TUM1166 Xe_Usp44 C283A, fl pQE80-‐F/A 6xhis E. Hörmanseder
TUM1245 Xe_UbcX wt,fl pQE80-‐F/A 6xhis E. Hörmanseder
UbcX dissociates XErp1 from the APC/C Material and Methods
57 5.2.3. Cloning and Mutagenesis
Full-‐length H.s. UbcH10, X.l. UbcX, X.l. USP44 and X.l. Cdc20 were amplified from cDNA libraries using primers described (Table 5.2.) and subcloned into a the corresponding vectors (Table 5.1.). Standard molecular biology techniques have been used to generate, clone and subclone DNA encoding the genes of interest. Plasmid DNA was u purified from the E. coli strain TG1 using the Qiagen Mini kit according to the manufacturers instructions. DNA fragments were isolated from agarose gels using the Qiagen gel elution kit. Restriction digests were carried out as recommended by the enzyme supplier (New England Biolabs). Polymerase chain reaction (PCR) was carried out using Pfu Turbo polymerase (Stratagene). Ligation reactions were done overnight at 18
°C with T4 ligase (Roche). Catalytically inactive UbcH10ci and UbcXci were generated by mutating cystein 114 of UbcH10wt and cystein 114 of UbcXwt respectively, to serine using the QuikChange kit (Stratagene) according to the manufacturer’s protocol.
5.3. Proteins
5.3.1. His-‐ tagged protein expression in bacteria
Bacteria of the E. coli strain JM109RIL were transformed with a plasmid encoding for the 6xhis -‐tagged Ubiquitinwt , UbiquitinΔGG, UbcH10wt, UbcH10ci, UbcXwt and UbcXci and an overnight culture was grown in 100 ml 1x LB (Roth) containing 100µg/ml ampicillin, 34µg/ml chloramphenicol and expanded to a 2L culture. When the culture reached an OD600=0,6, protein expression was induced by adding IPTG to a final concentration of 1 mM. The induction period was 4 hours at 37°C, after which the bacteria were harvested by centrifugation at 6000xg for 10 minutes at 4°C and the bacterial pellets were snap frozen in liquid nitrogen and stored at -‐80°C.
UbcX dissociates XErp1 from the APC/C Material and Methods
5.3.2. His-‐tagged protein expression in SF9 cells
SF9 insect cells were cultured at 27 °C in TC-‐100 medium (Gibco or PAN) supplemented with 10 % fetal calf serum (FCS) and antibiotics (1000 units/ml penicillin G and 1 mg/ml streptomycin). For protein expression, SF9 cells were seeded on 15 cm culture dishes at a density of 2x107 cells per dish and infected with the Cdc20 encoding baculovirus, which was generated according to the Bac-‐to-‐Bac protocol provided by Invitrogen. After 48h the cells were resuspended in culture medium and collected by centrifugation at 400xg for 10 min. The cell-‐pellet was snap frozen in liquid nitrogen and stored at -‐80°C.
5.3.3. His-‐tagged protein purification from bacteria and SF9 cells
Cell pellets were thawed at room temperature in 10 ml IMAC5 lysis buffer [5mM Imidazol, 300mM NaCl, 0.1%Triton X-‐100, 1x complete protease inhibitors (Roche), 20mM Tris-‐HCl pH 8.0] per liter of original bacterial culture or 500 ml of SF9 cell suspension, respectively. Cells were lysed with an Emulsi-‐
Flex-‐C5 and the lysate was cleared by centrifugation at 40.000 g for 15 minutes at 4.°C. His-‐tagged proteins were isolated by incubating the cleared lysate with 0,25 ml of NTA-‐ or NDA-‐agarose (Quiagen or Macherey-‐Nagel, respectively) per liter of original culture for 4 h at 4°C. The resin was washed two times in batch with 20 ml IMAC20(TA) [20mM Imidazol, 300mM NaCl, 1mM NaATP, 1mM MgCl2, 0.1%Triton X-‐100, 1x complete protease inhibitors (Roche), 20mM Tris-‐
HCl pH 8.0] and once with 20 ml of IMAC20 without ATP for 15 min at 4°C; the beads were collected by centrifugation at 1000xg for 5min at 4°C. The beads were transferred into a column (15 ml, Biorad). Proteins were eluated with one fraction of 0,25 ml and five fractions of 0,5 ml IMAC200 [200mM Imidazol, 300mM NaCl, 0.1%Triton X-‐100, 1x complete protease inhibitors (Roche), 20mM Tris-‐HCl pH 8.0]. The protein-‐containing fractions were dialyzed overnight at 4°C into an appropriate storage buffer [150 mM KCl, 1mM DTT, 10% Glycerol, 20 mM K-‐HEPES pH7.7]. Finally, protein aliquots were snap
UbcX dissociates XErp1 from the APC/C Material and Methods
59 frozen in liquid nitrogen and stored at -‐80°C. When the protein-‐solutions were thawed again, the samples were centrifuged for 10 min at 186.000xg to remove potential protein precipitates and the protein concentration was measured by standard Bradford assays.
5.3.4. Coupled in vitro transcription/translation (IVT)
IVT reactions to make 35S-‐labelled proteins were carried out using the TNT kit (Promega) in wheat-‐germ extract according to the manufacturer’s instructions, except 1 µg of plasmid DNA encoding the protein of interest and S35-‐labelled methionine was used and the reaction was incubated at 30°C for 2 hours. The successful translation of the protein was analyzed by SDS-‐PAGE and autoradiographic analysis.
5.4. Antibodies
5.4.1. Antibodies used in this study:
Antibodies against Cdc20 and cyclin B2 were purchased from Abcam, against Cdc27 and α-‐tubulin from Sigma. Antibodies against βTRCP were a kind gift of T. Lorca. Anti-‐XErp1 antibodies were described previously (Schmidt et al, 2005). Anti-‐UbcX antibodies were generated by immunizing rabbits with full-‐
length 6xhis-‐UbcX and affinity purification of the anti-‐UbcX antibodies from the obtained serum. Anti-‐USP44 antibodies were generated by immunizing two rabbits with a mix of USP44-‐232 peptide (KMNQKNSPTTKQKTPAPTSDKAC) and USP44-‐CT peptide (C-‐ENGHLSDTLPVHGSPQSPPR). Antibodies were affinity purified using 6xhis-‐USP441-‐200, USP44-‐232 peptide or USP44-‐CT peptide.
5.4.2. Affinity purification of antibodies
SulfoLink coupling resin (Pierce) was equilibrated at room temperature and 1,5 ml resin was transferred into a 15 ml plastic column (Biorad). The column was
UbcX dissociates XErp1 from the APC/C Material and Methods
washed with 10 ml of coupling buffer [5mM EDTA-‐Na, 50mM Tris pH8] and 1.5-‐
2 mg antigen with a concentration of 1mg/ml was applied. The resin was incubated with the antigen for 30 min at room temperature by end-‐over-‐end mixing. The column was put upright for 30min at room temperature. The antigen-‐coupled resin was washed with 6 ml coupling buffer. Nonspecific binding sites on the resin were blocked by the addition of 4 ml quenching buffer [50mM L-‐cystein, 50mM Tris, 5mM EDTA-‐Na, pH8] to the column. After 45 min incubation at room temperature, the column was washed with at least 6x 10 ml of wash solution [1M NaCl, 50mM Tris, 5mM EDTA-‐Na, pH8]. After this procedure, the ligand is covalently coupled to the resin and the column can be used for affinity purifications. If needed, the column was stored at 4°C in storage buffer [1x PBS, 0,05% NaN3].
For antibody affinity-‐purification, the column was washed with 10 ml 1x TBS, 10 ml elution buffer [150 mM NaCl, 200mM glycine pH 2.3] and TBS until the flow through has a pH=7,5. The serum (2 ml of anti-‐UbcX serum, 5 ml for anti-‐
USP44 serum) was filtered shortly before it was applied to the column through a 0,45mm filter. 1x TBS was added to the serum to reach a final volume of 10 ml and the solution was incubated with the column over night at 4°C. The column was washed with 3x 10 ml washing buffer [500mM NaCl, 0.2% Triton X100, 20mM Tris pH 7.5] and with 3x 10 ml TBS. The antibodies were eluted with 10x 500 ml elution buffer into tubes provided with 50-‐100 ml (determine volume empirically) Tris pH 8.5 to reach pH 7.5 after elution. The column was washed with 6x10 ml TBS and store in column storage buffer. The antibody eluates were dialyzed against 1xPBS containing 10% Glycerol, aliquoted and snap-‐frozen at -‐80°C.