PMC:7795856 / 7542-9075 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7795856","sourcedb":"PMC","sourceid":"7795856","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7795856","text":"2.1. Cloning and Bacterial Production of PASylated Tα1\nTo achieve C-terminal PASylation of N-terminally acetylated Tα1, a plasmid harboring a bicistronic operon was constructed to allow the simultaneous expression of human Tα1 (UniProtKB ID: P06454; residues 2–29), C-terminally fused with a PAS polypeptide comprising 601 amino acids [34], and the E. coli N-acetyltransferase RimJ (UniProtKB ID: P0A948) based on the vector pASK75 (Figure 1) [36]. In a parallel attempt, a plasmid encoding an N-terminally PASylated Tα1 was constructed, again, using plasmid pASK75 as the backbone (this time omitting the RimJ cistron, see below). Cytoplasmic gene expression was performed in both cases on a 2 L shake flask scale using the E. coli strain NEBexpress under control of the chemically inducible tet promoter/operator [36].\nThe whole cell lysate was analyzed prior to and 15 h after induction by Western blotting. Using a monoclonal antibody that recognizes an epitope of the PAS#1 sequence, a distinct band with an approximate molecular size above 250 kDa was detected (Figure 1b), which demonstrated successful bacterial expression of the full-length C-terminally PASylated Tα1 (Tα1-PAS) without signs of degradation. Of note, the unusually slow migration of Tα1-PAS (52.7 kD) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is well known for PASylated proteins [31,37] and can be explained by the poor binding of SDS (which provides the electrophoretic driving force) to the strongly hydrophilic PAS sequence.","divisions":[{"label":"title","span":{"begin":0,"end":54}},{"label":"p","span":{"begin":55,"end":820}}],"tracks":[{"project":"LitCovid-PubTator","denotations":[{"id":"175","span":{"begin":0,"end":3},"obj":"Gene"},{"id":"183","span":{"begin":115,"end":118},"obj":"Gene"},{"id":"184","span":{"begin":223,"end":226},"obj":"Gene"},{"id":"185","span":{"begin":517,"end":520},"obj":"Gene"},{"id":"186","span":{"begin":217,"end":222},"obj":"Species"},{"id":"187","span":{"begin":349,"end":356},"obj":"Species"},{"id":"188","span":{"begin":725,"end":732},"obj":"Species"},{"id":"189","span":{"begin":793,"end":796},"obj":"Chemical"},{"id":"195","span":{"begin":1173,"end":1176},"obj":"Gene"},{"id":"196","span":{"begin":1279,"end":1301},"obj":"Chemical"},{"id":"197","span":{"begin":1302,"end":1316},"obj":"Chemical"},{"id":"198","span":{"begin":1338,"end":1341},"obj":"Chemical"},{"id":"199","span":{"begin":1437,"end":1440},"obj":"Chemical"}],"attributes":[{"id":"A175","pred":"tao:has_database_id","subj":"175","obj":"Gene:6700"},{"id":"A183","pred":"tao:has_database_id","subj":"183","obj":"Gene:134864"},{"id":"A184","pred":"tao:has_database_id","subj":"184","obj":"Gene:134864"},{"id":"A185","pred":"tao:has_database_id","subj":"185","obj":"Gene:134864"},{"id":"A186","pred":"tao:has_database_id","subj":"186","obj":"Tax:9606"},{"id":"A187","pred":"tao:has_database_id","subj":"187","obj":"Tax:562"},{"id":"A188","pred":"tao:has_database_id","subj":"188","obj":"Tax:562"},{"id":"A189","pred":"tao:has_database_id","subj":"189","obj":"MESH:C010349"},{"id":"A195","pred":"tao:has_database_id","subj":"195","obj":"Gene:134864"},{"id":"A196","pred":"tao:has_database_id","subj":"196","obj":"MESH:D012967"},{"id":"A197","pred":"tao:has_database_id","subj":"197","obj":"MESH:C016679"},{"id":"A198","pred":"tao:has_database_id","subj":"198","obj":"MESH:D012967"},{"id":"A199","pred":"tao:has_database_id","subj":"199","obj":"MESH:D012967"},{"subj":"175","pred":"source","obj":"LitCovid-PubTator"},{"subj":"183","pred":"source","obj":"LitCovid-PubTator"},{"subj":"184","pred":"source","obj":"LitCovid-PubTator"},{"subj":"185","pred":"source","obj":"LitCovid-PubTator"},{"subj":"186","pred":"source","obj":"LitCovid-PubTator"},{"subj":"187","pred":"source","obj":"LitCovid-PubTator"},{"subj":"188","pred":"source","obj":"LitCovid-PubTator"},{"subj":"189","pred":"source","obj":"LitCovid-PubTator"},{"subj":"195","pred":"source","obj":"LitCovid-PubTator"},{"subj":"196","pred":"source","obj":"LitCovid-PubTator"},{"subj":"197","pred":"source","obj":"LitCovid-PubTator"},{"subj":"198","pred":"source","obj":"LitCovid-PubTator"},{"subj":"199","pred":"source","obj":"LitCovid-PubTator"}],"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_"}]},{"project":"LitCovid-sentences","denotations":[{"id":"T48","span":{"begin":0,"end":4},"obj":"Sentence"},{"id":"T49","span":{"begin":5,"end":54},"obj":"Sentence"},{"id":"T50","span":{"begin":55,"end":448},"obj":"Sentence"},{"id":"T51","span":{"begin":449,"end":631},"obj":"Sentence"},{"id":"T52","span":{"begin":632,"end":820},"obj":"Sentence"},{"id":"T53","span":{"begin":821,"end":910},"obj":"Sentence"},{"id":"T54","span":{"begin":911,"end":1216},"obj":"Sentence"},{"id":"T55","span":{"begin":1217,"end":1533},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"attributes":[{"subj":"T48","pred":"source","obj":"LitCovid-sentences"},{"subj":"T49","pred":"source","obj":"LitCovid-sentences"},{"subj":"T50","pred":"source","obj":"LitCovid-sentences"},{"subj":"T51","pred":"source","obj":"LitCovid-sentences"},{"subj":"T52","pred":"source","obj":"LitCovid-sentences"},{"subj":"T53","pred":"source","obj":"LitCovid-sentences"},{"subj":"T54","pred":"source","obj":"LitCovid-sentences"},{"subj":"T55","pred":"source","obj":"LitCovid-sentences"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"LitCovid-PubTator","color":"#93ec9e","default":true},{"id":"LitCovid-sentences","color":"#ec93a1"}]}]}}