TAP purification

Essentially, the purification was done according to Puig et al. (2001).

4.2.8.1. Cell harvest and lysis

A 2 L culture of yeast cells was harvested at an OD600 of 3,0 – 3,5 by centrifugation at 4000 rpm (SLC6000 rotor) for 4 min, washed once with 500 ml H2O and once with TAP buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1.5 mM MgCl2, 0,15% NP40). Cells were then flash frozen in liquid nitrogen and stored at -80°C.

For lysis, cells were resuspended in an equal volume of TAP buffer (containing 1mM DTT and protease inhibitors – 1.3 µg/ml pepstatin A, 0.28µg/ml leupeptin, 170 µg/ml PMSF, 330 µg/ml benzamidine) and mixed with a double volume of glass beads. Lysis occurred while shaking in a bead mill (Fritsch, Idar-Oberstein) by following this protocol: 3 x (500rpm for 4 min, 2 min breaks in between). The glass beads were removed and washed once with buffer, so that the lysate comprised 25 ml. After centrifugation at 4000 rpm (Rotanda 46R centrifuge) at 4°C for 10 min, the supernatant was subjected to ultracentrifugation (27000 rpm – SW32 rotor, 4°C, 1 h). The fatty phase was removed by aspiration and the clear lysate collected. It was either used directly or glycerol (final concentration of 5%) was added for flash freezing in liquid nitrogen and storage at -80°C.

4.2.8.2. Purification

0,4 ml IgG sepharose (GE Healthcare, Munich) were washed 3 x in TAP buffer (1800 rpm (Rotanda 46R centrifuge), 4°C, 2 min) and added to the lysate. After incubation for at least 1 h and up to overnight incubation on a turning wheel at 4°C, the beads were centrifuged down and transferred to a mobicol column containing a 35 µm filter. The beads were washed with 10 ml TAP buffer containing 0.5 mM DTT by gravity flow.

To elute the protein (complex), 4µl of TEV protease (0.2 – 10 µg/µl) and 150 µl TAP buffer (+ 0.5 mM DTT) were added to the beads and incubated on a turning wheel at 16°C for 1 h 20 min. For elution, the mobicol was centrifuged in a table top centrifuge at 2000 rpm for 1 min. The eluate was mixed with glycerol to a final concentration of 5-10%, flash frozen and stored at -80°C.

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4.2.8.3. Specific modifications for purification

A: Purification of the CTDK-I complex

12 L culture of SDC-URA-Leu media were inoculated with 72 OD600 (approx. 15 ml saturated culture) of the strain CTK1-TAP (RS453) containing the plasmids pRS425-CTK2-CTK3 and pRS426-CTK1-TAP. The cells were harvested and lysed according to the normal TAP purification protocol (4.2.8.1). The lysate was directly incubated with the IgG beads overnight. The beads were washed with 8 ml TAP buffer containing 1M NaCl and 0.5 mM DTT, followed by 5 ml of normal TAP buffer (+ 1 mM DTT). The TEV cleavage and IgG elution was done according to standard methods (4.2.8.2). During TEV cleavage, 0.5 ml calmodulin beads (GE Healthcare, Munich) were washed 3 x in TAP buffer plus 1 mM DTT and 2 mM CaCl2. After removal of surplus buffer, the beads were mixed with 150µl TAP buffer containing 1 mM DTT and 4 mM CaCl2 and incubated with 150µl TEV eluate at 4°C on a turning wheel for at least 1 h. The beads were washed with 5 ml TAP buffer plus 1 mM DTT and 2 mM CaCl2.

To elute the proteins, the beads were incubated in a thermomixer at 10°C, 650 rpm for 2 h with elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl 10 mM EGTA, 1mM DTT, Protease inhibitors) and then eluted by centrifugation in a tabletop centrifuge at 2000 rpm for 1 min. The eluate was concentrated using a 100 kDa MembraSpin Mini (membraPure, Bodenheim) filter until a volume of approximately 100 µl. Glycerol was added to a final concentration of 5-10 % and the eluate flash frozen in liquid nitrogen and stored at -80°C. B: Purification of candidate substrates for in vitro kinase assays

For purification of candidate substrates of Ctk1, the corresponding TAP tagged strains were cultured, harvested and lysed according to the standard protocol (4.2.8.1). When protein complexes were purified, the standard purification protocol was used (4.2.8.2). When single proteins were purified, a high salt washing step with 10 ml TAP buffer containing 1 M NaCl was included during the washing of the IgG beads, followed by a 2 ml wash with normal TAP buffer. After elution from the IgG beads, the eluate was concentrated to approximately 10-20 µl using a MembraSpin Mini concentrator (10 kDa; 30 kDa or 100 kDa cutoff; membraPure, Bodenheim). Glycerol was added to a final concentration of 5-10 % and the eluate flash frozen in liquid nitrogen and stored at -80°C.

C: Small scale TAP purification

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cells harvested by centrifugation (3600 rpm – Rotanda 46R centrifuge, 3 min, 4°C). The pellet was washed with 1 ml of cold H2O and finally resuspended in 1 ml cold TAP buffer containing 1 M NaCl, protease inhibitors (1.3 µg/ml pepstatin A, 0.28µg/ml leupeptin, 170 µg/ml PMSF, 330 µg/ml benzamidine), phosphatase inhibitors (10 mM NaF, 1 mM NaVO4, 5

mM β-glycerophosphate) and 1 mM DTT. For lysis, 1 ml of glass beads were added and the samples vortexed 5 x at RT for 3 min with 3 min breaks on ice. The lysate was collected by piercing of the reaction tube with a syringe and subsequent centrifugation (1800 rpm – Rotanda 46R centrifuge, 2 min, 4°C). The lysate was cleared by centrifugation at 13000 rpm in a table top centrifuge and 4°C for 30 min. 900µl of the supernatant were mixed with 50 µl pre-washed IgG beads and incubated on a turning wheel at 4°C for 1 h. The beads were then washed 10 x with TAP buffer containing 1 M NaCl, protease inhibitors and phosphatase inhibitors and 1 mM DTT by centrifugation at 2000 rpm (table top centrifuge) and 4°C for 2 min each. After the final washing step, 150 µl of TAP buffer (plus phosphatase inhibitors and 1 mM DTT) and 0.5 µl TEV protease (0.2 – 10 µg/µl) were added to the beads and the sample incubated at 16°C for 1 h 30 min. The TEV eluate was collected by centrifugation (1800 rpm – Rotanda 46R centrifuge, RT, 2 min) and precipitated as well as desalted according to the sample preparation and electrophoresis section of the Pro-Q® Diamond Phosphostain Gel Stain manual (Molecular Probes, Invitrogen, Karlsruhe).