Cell disrupter

The cells were harvested using a Beckman centrifuge equipped with the JLA 8.1000 rotor at 7,500 rpm (~10,000 × g) for 30 min. The cells were homogenized in 4 ml cell lysis buffer (100 mM Tris-HCl pH 7.5, 5 mM MgSO₄, some crystals phenylmethanesulfonyl fluoride, some crystals DNaseI (Roche)) per 1 g of cells (wet weight), and passed through a cell disrupter 4 times at 40 kPsi (Constant Systems). Cell debris was collected by centrifugation at 27,000 × g for 20 min. (Type 45Ti rotor, Beckman) and subsequently the membranes were collected by ultracentrifugation at 150,000 × g for 90 min (Type 45Ti rotor, Beckman).

hMLC was cloned into the His-bsSumo (K151) expression vector using Transfer-PCR. An Ala residue was incorporated at the N-terminal part of hMLC in order to facilitate cleavage of hMLC from the His-bdSumo tag. Expression was performed in E coli Bl21(DE3) cells. Protein production was induced by addition of 0.2 mM IPTG at mid-log phase and the cells were grown overnight at 15 °C. Cells were lysed using a cell disrupter (Constant Systems) at 4 °C in buffer A (50 mM Tris pH 8, 0.5 M NaCl, 20 mM Imidazole with EDTA-free protease-inhibitor tablet, 0.5 mg/ml lysozyme, and DNaseI). After centrifugation, the clarified supernatant was passed over a HiTrap chelating HP 5 ml column (GE, Healthecare). 1 ml bdSumo protease, bdSENP1, without His tag was added to the column which was left at 4 °C overnight. The cleaved protein was collected by washing the column with 10 ml buffer A. hMLC was further purified by size-exclusion chromatography (HiLoad_16/60_superdex75 prepgrade, GE Healthcare) equilibrated with 50 mM Tris pH 8, 0.1 M NaCl.
DAPK2 K42A and K42A S289A were cloned into the pET28-TevH expression vector using Transfer-PCR. Plasmids were expressed in Escherichia coli BL21(DE3) cells. Proteins were purified using excess peptide.

HI18 was selected from a β-wrapin phage display library as described17 (link). Protein expression was carried out using a pET302/NT-His vector encoding HI18 with an N-terminal His₆-tag in E. coli BL21(DE3) cells in LB medium. Expression was induced with 1 mM IPTG at OD 0.6–0.8 followed by overnight incubation at 27 °C. The cell pellet was resuspended in 20 mM imidazole, 500 mM NaCl, 20 mM sodium phosphate, pH 8.0, containing EDTA-free protease inhibitors (Roche Applied Sciences) and lysed by a cell disrupter (Constant Systems). Insoluble material was removed by centrifugation at 28,000 × g and the supernatant was loaded on a HisTrap FF 5 ml column (GE Healthcare). The dimeric form of HI18 was collected from a HiLoad 16/60 Superdex 75 size-exclusion chromatography column (GE Healthcare) in 20 mM sodium phosphate, pH 6.0. For NMR experiments, HI18 was expressed in M9 minimal medium supplemented with ¹⁵N-NH₄Cl (1 g/l) and ¹³C₆-glucose (2 g/l) and purified as above.

In order to produce a specific antibody against ADO, the ado gene (sll0208) with additional 6x His-tag at the N-terminal end was amplified from genomic DNA and subcloned into a commercial pACYC plasmid (Novagen). The pACYC-sll0208 construct was confirmed by sequencing (MWG) and used for protein over-expression in E. coli BL21. Protein production was induced with 0.5mM isopropyl-β-d-thiogalactopyranoside (IPTG), and after ~16h, the cells were lysed with Cell Disrupter (Constant Systems Ltd) using 20 kpsi and the cell debris was removed. To purify the His-tagged ADO, the supernatant was mixed with 1ml pretreated TALON metal affinity resin and the bound protein was eluted with 20mM sodium phosphate pH 7, 100mM NaCl, 250mM imidazole, and 10% glycerol. The elution fraction with highest protein concentration was identified with Bradford reagent, and the target protein of ADO corresponding ~27kDa was separated with SDS-PAGE (Mini-PROTEAN^® TGX^™, Sigma) and sent to Agrisera to raise a polyclonal antibody against ADO. The function of the antibody was verified in Western blot using different concentrations of fractionated and unfractionated cell samples.

Cells were grown overnight in 50 ml flasks in SD-URA medium at 30°C. After growth cells were inoculated to a cell density of 1 × 10⁷ cells/ml into 1000 ml SG-URA and induction was completed by addition of 1000 μl of a 1 mM ES stock solution. Induction was performed at 25°C for 20 h. Induction of GOase-tGFP expression was checked by flow cytometry. The cells were precipitated and the supernatant was concentrated to 25 ml using a Vivaflow 200, 10.000 MWCO Hydrosart (Sartorius) laboratory crossflow cassette. The cell pellet was resuspended in PBS, disrupted using a cell disrupter (Constant systems LTD) and cell debris was removed by centrifugation. Both supernatant and cell debris were analyzed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as ABTS assay. SDS-PAGE was performed as described in (19 ).