PMC:5115869 / 51543-56670 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/5115869","sourcedb":"PMC","sourceid":"5115869","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5115869","text":"Protein expression and purification\nFor co-expression of biotin ligase and HCN2 constructs (monomeric or tetrameric versions), BL21 CodonPlus(DE3)-RIL cells were sequentially transformed first with the HCN2 construct, selecting transformants overnight on kanamycin/chloramphenicol plates, then with BirA construct, selecting overnight on ampicillin/kanamycin/chloramphenicol plates. Several clones were picked to inoculate 125 ml of MDG medium (Studier, 2005) and cultured overnight at 37°C. 30 ml of the resulting culture was used to inoculate 1 L of LB medium in 2 L shake flasks that were grown at 37°C until OD600 of about 0.5 (all OD600 values refer to measurements done in Beckman DU-640 spectrophotometer), at which point 1 ml of 100 mM solution of biotin in DMSO was added to each flask. After additional 30 min of shaking, the cultures were cooled on ice and induced with 1 mM IPTG. After 20 hr of growth at 16°C, cells from 4 L of culture were pelleted, washed in 1 L of ice-cold 20 mM Tris, 100 mM NaCl and 2 mM EDTA, pH 8.0, the cell paste frozen in liquid nitrogen and stored at −80°C until needed. Expression of the construct for crystallization followed the same outline except that seed culture used was grown at 30°C overnight in MDG with kanamycin/chloramphenicol and no biotin was added before induction at OD600 1.0.\nThe biotinylated HCN2 constructs containing the entire C-linker sequences were purified as follows. Unless otherwise stated, all procedures were performed at 4°C. 10 g of frozen cells were resuspended in 60 ml of buffer A (20 mM HEPES, 200 mM NaCl, 25 mM imidazole, 0.5 mM TCEP, 10% v/v glycerol, pH 7.5) with an addition of extra 0.5 mM TCEP and protease inhibitors (house-made cocktail equivalent to Roche’s 'cOmplete EDTA-free' tablets). The cells were disrupted with ten cycles of sonication on ice-water bath at ~93 W power output while monitoring suspension temperature, keeping cycles short enough to prevent temperature raising above 8°C and resuming at 2–3°C. The suspension was spun for 30 min at 48,000 g and the supernatant was loaded by gravity onto a 6 ml Ni-NTA (Qiagen) equilibrated with buffer A. The column was then washed by gravity with 200 ml of buffer A followed by two 50 ml wash steps with modified buffer A containing higher final imidazole concentrations, 32 and 40 mM. Remaining bound protein was eluted with 18 ml of modified buffer A containing 250 mM imidazole. Approximately 1 mg of TEV protease per 50 mg of eluted protein was added and the mixture was dialyzed overnight against 2 L of 20 mM HEPES, 100 mM NaCl, 0.5 mM TCEP, pH 7.5 in dialysis bags with 15 kDa cut-off (Spectrum Laboratories cat. # 132122). Following dialysis, a precipitate that formed in the case of mutant HCN2s was spun down for 20 min at 48,000 g and the supernatant loaded by gravity onto a fresh 10 ml Ni-NTA column equilibrated with 20 mM HEPES, 200 mM NaCl, 0.5 mM TCEP, 10% glycerol, pH 7.5. After 100 ml wash with the same buffer that removes minor contaminants, the untagged HCN2 was eluted isocratically with 10 mM imidazole in the equilibration buffer. The eluted protein was concentrated by ultrafiltration (Amicon Ultra-15, 10 kDa cut-off) at room temperature to approximately 7 mg/ml and frozen in liquid nitrogen as 0.3 ml aliquots in screw cap tubes.\nPurification of the MBP-HCN2 fusion for crystallization followed the same protocol as for biotinylated HCN2 with the following exceptions: purification used 15 g of cells and 100 ml of buffer A; ethylene glycol was used in place of glycerol in buffer A; first Ni-NTA column was 10 ml in volume and the second column was 13 ml; the buffer used during the second Ni-NTA contained 100 mM NaCl and no glycerol. The final protein fraction was concentrated by ultrafiltration (Amicon Ultra-15, 50 kDa cut-off) to approximately 140 mg/ml, frozen in liquid nitrogen as droplets of about 30 µl and stored at –80°C until needed.\nPurification of the GCN4pLI-HCN2 tetramer followed identical steps to the monomer up to the elution from the first Ni-NTA column. Thereafter, the eluate (20 ml) was dialyzed against 20 mM HEPES, 100 mM NaCl, 1 mM TCEP, 0.1 mM EDTA, pH 7.5 overnight and dialyzate mixed with an equal volume of 40 mM HEPES, 600 mM NaCl, 20% glycerol, 1 mM TCEP, pH 7.5 before adding ~1:20 of TEV protease by protein mass for cleavage overnight at 4°C. Precipitated HCN2 tetramer was separated from MBP by centrifugation at 2500 g for 15 min and the pellet resuspended in 40 mM HEPES, 600 mM NaCl, 20% glycerol, 2 mM TCEP, 0.1% LDAO (Sigma, cat. no. 40236), pH 7.5 (Buffer B), followed by homogenization in glass-teflon Potter-Elvehjem homogenizer. Following 30 min centrifugation at 40,000 g, the solubilized fraction was loaded onto a fresh Ni-NTA column and the GCN4pLI-GCN2 fusion eluted in the Buffer B containing 20 mM imidazole. The eluate (~13 mg/ml protein) was flash-frozen in liquid nitrogen as 0.6 ml aliquots and stored at –80°C until needed.\nIn all cases, protein concentration was determined from A280 using theoretical extinction coefficient values derived by the Protparam tool (Gasteiger et al., 2005).","divisions":[{"label":"title","span":{"begin":0,"end":35}},{"label":"p","span":{"begin":36,"end":1336}},{"label":"p","span":{"begin":1337,"end":3306}},{"label":"p","span":{"begin":3307,"end":3925}},{"label":"p","span":{"begin":3926,"end":4962}}],"tracks":[{"project":"2_test","denotations":[{"id":"27858593-15915565-27104482","span":{"begin":454,"end":458},"obj":"15915565"}],"attributes":[{"subj":"27858593-15915565-27104482","pred":"source","obj":"2_test"}]},{"project":"MyTest","denotations":[{"id":"27858593-15915565-27104482","span":{"begin":454,"end":458},"obj":"15915565"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"attributes":[{"subj":"27858593-15915565-27104482","pred":"source","obj":"MyTest"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#a493ec","default":true},{"id":"MyTest","color":"#9cec93"}]}]}}