PMC:7795856 / 25823-28012 JSONTXT

{"project":"LitCovid-PubTator","denotations":[{"id":"465","span":{"begin":825,"end":828},"obj":"Gene"},{"id":"466","span":{"begin":840,"end":843},"obj":"Gene"},{"id":"467","span":{"begin":1015,"end":1018},"obj":"Gene"},{"id":"468","span":{"begin":1269,"end":1272},"obj":"Gene"},{"id":"469","span":{"begin":1538,"end":1541},"obj":"Gene"},{"id":"470","span":{"begin":1648,"end":1651},"obj":"Gene"},{"id":"471","span":{"begin":82,"end":93},"obj":"Chemical"},{"id":"472","span":{"begin":357,"end":373},"obj":"Chemical"},{"id":"473","span":{"begin":512,"end":522},"obj":"Chemical"},{"id":"474","span":{"begin":524,"end":528},"obj":"Chemical"},{"id":"475","span":{"begin":536,"end":544},"obj":"Chemical"},{"id":"476","span":{"begin":551,"end":555},"obj":"Chemical"},{"id":"477","span":{"begin":633,"end":637},"obj":"Chemical"},{"id":"478","span":{"begin":960,"end":971},"obj":"Chemical"},{"id":"479","span":{"begin":1085,"end":1095},"obj":"Chemical"},{"id":"480","span":{"begin":1103,"end":1113},"obj":"Chemical"},{"id":"481","span":{"begin":1182,"end":1199},"obj":"Chemical"},{"id":"482","span":{"begin":1253,"end":1256},"obj":"Chemical"},{"id":"483","span":{"begin":1348,"end":1352},"obj":"Chemical"},{"id":"484","span":{"begin":1604,"end":1608},"obj":"Chemical"},{"id":"485","span":{"begin":1714,"end":1724},"obj":"Chemical"},{"id":"486","span":{"begin":1732,"end":1744},"obj":"Chemical"},{"id":"487","span":{"begin":1745,"end":1748},"obj":"Chemical"},{"id":"488","span":{"begin":1893,"end":1903},"obj":"Chemical"},{"id":"489","span":{"begin":1966,"end":1970},"obj":"Chemical"},{"id":"490","span":{"begin":2070,"end":2078},"obj":"Chemical"},{"id":"491","span":{"begin":2130,"end":2135},"obj":"Chemical"},{"id":"492","span":{"begin":103,"end":116},"obj":"Disease"},{"id":"493","span":{"begin":1783,"end":1805},"obj":"Disease"}],"attributes":[{"id":"A465","pred":"tao:has_database_id","subj":"465","obj":"Gene:134864"},{"id":"A466","pred":"tao:has_database_id","subj":"466","obj":"Gene:134864"},{"id":"A467","pred":"tao:has_database_id","subj":"467","obj":"Gene:134864"},{"id":"A468","pred":"tao:has_database_id","subj":"468","obj":"Gene:134864"},{"id":"A469","pred":"tao:has_database_id","subj":"469","obj":"Gene:134864"},{"id":"A470","pred":"tao:has_database_id","subj":"470","obj":"Gene:134864"},{"id":"A471","pred":"tao:has_database_id","subj":"471","obj":"MESH:D019343"},{"id":"A472","pred":"tao:has_database_id","subj":"472","obj":"MESH:D000645"},{"id":"A476","pred":"tao:has_database_id","subj":"476","obj":"MESH:D004492"},{"id":"A483","pred":"tao:has_database_id","subj":"483","obj":"MESH:D012965"},{"id":"A484","pred":"tao:has_database_id","subj":"484","obj":"MESH:D012965"},{"id":"A486","pred":"tao:has_database_id","subj":"486","obj":"MESH:D019856"},{"id":"A487","pred":"tao:has_database_id","subj":"487","obj":"MESH:D006851"},{"id":"A489","pred":"tao:has_database_id","subj":"489","obj":"MESH:D012965"},{"id":"A490","pred":"tao:has_database_id","subj":"490","obj":"MESH:D010455"},{"id":"A491","pred":"tao:has_database_id","subj":"491","obj":"MESH:D014867"},{"id":"A492","pred":"tao:has_database_id","subj":"492","obj":"MESH:C537005"},{"id":"A493","pred":"tao:has_database_id","subj":"493","obj":"MESH:C562593"}],"namespaces":[{"prefix":"Tax","uri":"https://www.ncbi.nlm.nih.gov/taxonomy/"},{"prefix":"MESH","uri":"https://id.nlm.nih.gov/mesh/"},{"prefix":"Gene","uri":"https://www.ncbi.nlm.nih.gov/gene/"},{"prefix":"CVCL","uri":"https://web.expasy.org/cellosaurus/CVCL_"}],"text":"Bacteria were harvested by centrifugation and disrupted in the presence of 100 mM citric acid using an EmulsiFlex C5 cell homogenizer (Avestin, Mannheim, Germany). After centrifugation for 20 min (39,200× g) at 16 °C, the supernatant was filter-sterilized using a 0.45 µm syringe filter (Sartorius, Göttingen, Germany). The clear filtrate was adjusted with ammonium sulfate to 30% saturation, stirred for 30 min at RT, and centrifuged for 30 min (39,200× g) at RT. The precipitate was solubilized in subtractive AEX buffer (sAEX; 20 mM Tris/HCl, 1 mM EDTA, pH 8.5), filter-sterilized, and dialyzed twice against a 100-fold volume of sAEX buffer at 4 °C. Subsequent chromatography steps were performed on an ÄKTA Explorer 100 system (GE Healthcare, Freiburg, Germany) operated at a flow rate of 5 mL/min. The dialyzed sample (Tα1-PAS or PAS-Tα1) was first applied to a 5 mL TOYOPEARL NH2-750F column (Tosoh Bioscience, Griesheim, Germany) equilibrated with the sAEX buffer. The flow-through containing the PASylated Tα1 was collected and dialyzed twice against a 100-fold volume of the CEX buffer (20 mM Na-citrate, pH 3.0) at 4 °C. The protein solution was then applied to an 85 mL TOYOPEARL Sulfate-650F column (Tosoh Bioscience) equilibrated with the CEX buffer. PAS-Tα1 quantitatively bound to the column and the pure protein was eluted using a NaCl concentration gradient (0–250 mM over 2 column volumes). In the case of Tα1-PAS, the CEX flow-through fraction contained the N-terminally acetylated protein, whereas the non-acetylated Tα1-PAS stayed bound to the column and could again be eluted in a NaCl concentration gradient. The acetylated Tα1-PAS fraction from the flow-through was dialyzed twice against AEX buffer (20 mM ethanolamine/HCl, pH 10) and then applied to a 100 mL Fractogel® EMD TMAE (M)strong anion exchanger (Merck Millipore, Burlington, MA, USA) equilibrated with the AEX buffer. This time, highly pure acetylated Tα1-PAS was eluted using a NaCl concentration gradient (0–200 mM over 2 column volumes) in the AEX buffer. Both purified PASylated peptides were dialyzed 5 times against a 200-fold volume of water, lyophilized, and stored at −20 °C until further use."}

{"project":"LitCovid-sentences","denotations":[{"id":"T150","span":{"begin":0,"end":163},"obj":"Sentence"},{"id":"T151","span":{"begin":164,"end":319},"obj":"Sentence"},{"id":"T152","span":{"begin":320,"end":464},"obj":"Sentence"},{"id":"T153","span":{"begin":465,"end":653},"obj":"Sentence"},{"id":"T154","span":{"begin":654,"end":803},"obj":"Sentence"},{"id":"T155","span":{"begin":804,"end":972},"obj":"Sentence"},{"id":"T156","span":{"begin":973,"end":1131},"obj":"Sentence"},{"id":"T157","span":{"begin":1132,"end":1264},"obj":"Sentence"},{"id":"T158","span":{"begin":1265,"end":1409},"obj":"Sentence"},{"id":"T159","span":{"begin":1410,"end":1632},"obj":"Sentence"},{"id":"T160","span":{"begin":1633,"end":1904},"obj":"Sentence"},{"id":"T161","span":{"begin":1905,"end":2045},"obj":"Sentence"},{"id":"T162","span":{"begin":2046,"end":2189},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"Bacteria were harvested by centrifugation and disrupted in the presence of 100 mM citric acid using an EmulsiFlex C5 cell homogenizer (Avestin, Mannheim, Germany). After centrifugation for 20 min (39,200× g) at 16 °C, the supernatant was filter-sterilized using a 0.45 µm syringe filter (Sartorius, Göttingen, Germany). The clear filtrate was adjusted with ammonium sulfate to 30% saturation, stirred for 30 min at RT, and centrifuged for 30 min (39,200× g) at RT. The precipitate was solubilized in subtractive AEX buffer (sAEX; 20 mM Tris/HCl, 1 mM EDTA, pH 8.5), filter-sterilized, and dialyzed twice against a 100-fold volume of sAEX buffer at 4 °C. Subsequent chromatography steps were performed on an ÄKTA Explorer 100 system (GE Healthcare, Freiburg, Germany) operated at a flow rate of 5 mL/min. The dialyzed sample (Tα1-PAS or PAS-Tα1) was first applied to a 5 mL TOYOPEARL NH2-750F column (Tosoh Bioscience, Griesheim, Germany) equilibrated with the sAEX buffer. The flow-through containing the PASylated Tα1 was collected and dialyzed twice against a 100-fold volume of the CEX buffer (20 mM Na-citrate, pH 3.0) at 4 °C. The protein solution was then applied to an 85 mL TOYOPEARL Sulfate-650F column (Tosoh Bioscience) equilibrated with the CEX buffer. PAS-Tα1 quantitatively bound to the column and the pure protein was eluted using a NaCl concentration gradient (0–250 mM over 2 column volumes). In the case of Tα1-PAS, the CEX flow-through fraction contained the N-terminally acetylated protein, whereas the non-acetylated Tα1-PAS stayed bound to the column and could again be eluted in a NaCl concentration gradient. The acetylated Tα1-PAS fraction from the flow-through was dialyzed twice against AEX buffer (20 mM ethanolamine/HCl, pH 10) and then applied to a 100 mL Fractogel® EMD TMAE (M)strong anion exchanger (Merck Millipore, Burlington, MA, USA) equilibrated with the AEX buffer. This time, highly pure acetylated Tα1-PAS was eluted using a NaCl concentration gradient (0–200 mM over 2 column volumes) in the AEX buffer. Both purified PASylated peptides were dialyzed 5 times against a 200-fold volume of water, lyophilized, and stored at −20 °C until further use."}