The correct plasmids were RepSox sequenced and transformed into the respective yeast strains by electroporation [43]. Heterologous expression and purification of recombinant Pof1p: Recombinant Pof1p, which possesses an N-terminal His-tag, was expressed in the E. coli BL21 (DE3) strain that was transformed with the pET15b-Pof1p plasmid (the POF1 coding region was cloned into the expression vector pET15b from Novagen using the NdeI and BamHI restriction sites). The cells were cultured (50 mL) overnight in LB + ampicillin (100 μg/mL), transferred
to 1 L of fresh LB + ampicillin medium and cultured further until the OD600 nm reached 0.6-0.8. IPTG was added to a final concentration of 1 mM. After 3 h of incubation at 37°C, the cells were harvested by centrifugation. The pellet was washed and suspended in the start buffer composed of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl and 20 mM imidazole. The cells were sonicated twice for 45 s (40% amplitude),
followed by 30 s on ice between sonications using a Branson Cell Disruptor. The cell extracts were kept on ice during streptomycin sulfate treatment (1% for 20 min), and the suspension was centrifuged at 16,000 g for 30 min to Alpelisib supplier remove nucleic acid precipitates and cell debris. Finally, the extracts were applied to a Hi-trap nickel-affinity column (Life Technologies). The conditions for protein purification were optimized using the gradient procedure for imidazole concentration described by the manufacturer. Thin Layer see more Chromatography (TLC) analyses: The assays were performed as previously
described [44]. Briefly, the reaction media contained 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 20 μM phosphatidylcholine:oleate vesicles, 10 mM DTT, 1.5 mM phosphocholine (or 2 mM phosphoethanolamine), 1 μg/μL (20 μM) Pof1p and 200 μCi/μmol of [α-32P]CTP or [α-32P]ATP. The reactions were incubated at 37°C overnight in the presence of [α-32P]CTP or 2 h in the presence of [α-32P]ATP. Controls were subjected to the same conditions in the absence of Pof1p. The reactions were analyzed by TLC at room temperature using silica gel plates (Merck) with a solvent system composed of ethanol/NH4OH (1:1). The plates were autoradiographed, and the resulting bands were compared acetylcholine with [α-32P]CTP or [α-32P]ATP without any incubation or addition of enzyme. ATPase activity. The reactions containing 1 mM ATP, 1 μM Pof1p, 5 mM MgCl2 and 100 mM Tris-HCl (pH 7.5) were incubated at 37°C for 1 h. Subsequently, the reactions were boiled for 5 min and centrifuged for 10 min at 16,000 g. The PiPer Phosphate assay mix was added to the supernatant according to the manufacturer’s instructions (Molecular Probes – Invitrogen). The reactions were incubated at 37°C for an additional 1 h in the dark. The absorbance of resorufin, the Amplex Red reagent reaction product, was detected by its absorbance at 565 nm.
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