PMC:2728203 / 27104-28601 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"Residues 1–105 of Nab2 were cloned into pET28a and pET30a vectors (Novagen) via NdeI and BamHI sites to produce a His-tagged and untagged construct, respectively. Proteins were expressed in E. coli BL21 (DE3) CodonPlus RIL cells by overnight induction with 1 mM IPTG at 25 °C. His-tagged Nab2 1–105 was purified by using Ni–NTA agarose resin according to manufacturer's instructions (Qiagen Inc.) followed by gel filtration on a Superdex 75 HiLoad 26/60 prep grade column (GE Healthcare) with 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, and 50 mM NaCl as buffer. Untagged protein was purified by anion-exchange chromatography (Q-Sepharose HiLoad 16/10 column, GE Healthcare) applying in 20 mM Bis-Tris–HCl, pH 6.0, 1 mM DTT, and 1 mM EDTA and eluting with a linear gradient of NaCl from 0 to 500 mM in the same buffer followed by gel filtration chromatography as above. Typical yields were 40 mg/l of culture. The resultant proteins were \u003e 95% pure as assessed by SDS-PAGE. By mass spectrometry, the untagged material had an Mr of 11,324.6, consistent with Met1 having been removed (theoretical Mr = 11,455.2 for full length, with Mr = 11,324.0 corresponding to loss of the N-terminal Met), whereas the His-tagged construct had an Mr of 13,489 (theoretical Mr = 13487.2, corresponding to loss of the N-terminal Met). Nab2 dual labeled with 15N and 13C was purified from cultures harboring pET30a:Nab2 (1–105) grown in M9 minimal medium supplemented with 13C-labeled glucose and 15N-labeled NH4Cl.","tracks":[]}