Rosetta gami b de3 e coli

Chimeric EC2 fusion proteins were produced by overlap extension PCR, with the swapped regions described in S1 Table in S1 File. All of the wild-type and point mutant EC2 fusion proteins, except Y148M and Y148A, were previously described [25] (link); the Y148 mutants were produced in the same way. The production and characterisation of the tetraspanin EC2 GST fusion proteins has been described in detail previously [25] (link). Briefly, tetraspanin EC2 regions that had been cloned into the pGEX-KG vector were expressed in Rosetta Gami B(DE3) E. coli (Merck Biosciences), culturing at 37°C for 4 hours after IPTG induction. Cells were pelleted and lysed by sonication in the presence of a protease inhibitor cocktail. Recombinant protein was purified in a single step by affinity chromatography on glutathione beads (Amersham-Pharmacia). Protein purity was analysed by Coomassie staining of SDS-PAGE gels and total protein concentration determined by Bradford assay. As it was not possible to separate the full-length EC2 fusion protein from the smaller fragments produced, the percentage of full length material in each sample was measured by densitometry. This was plotted against the inhibitory activity of each sample to ensure that inhibition of MGC formation was not a simple function of the concentration of the full length fusion protein (S1 Fig. in S1 File).

FLAG-CTCF-6xHis (wild type, S224A, and S224E) and FLAG-GFP-6xHis were made as previously described in Rosetta-Gami B (DE3) E. coli (EMD Millipore) using pBD39, pBD40, pBD116, and pBD117 (Sun et al., 2013 (link)). 2.5 μg FLAG-CTCF-6xHis was in vitro phosphorylated with 250IU Casein Kinase 2 (CK2) (NEB, P6010) and 200 μM ATP (1 μCi [γ³²P] ATP) for 30 min at 30C. 1 μg FLAG-CTCF-6xHis and 0.5 μg dephosphorylated casein (SignalChem C03-54BN) were also in vitro phosphorylated with 0.1 μg Polo Like Kinase 1 (PLK1) (Sigma, P-0060) in 5 mM MOPS pH7.2, 2.5 mM β-glycerophosphate, 4 mM MgCl₂, 1 mM EGTA, 0.05 mM DTT, and 200 μM ATP (1 μCi [γ³²P] ATP) for 60 min at 30C. CK2 and PLK1 labeling was detected by SDS-PAGE and autoradiography. For mass spectrometry, FLAG-CTCF-6xHis was phosphorylated with non-radioactive ATP.