PMC:7795856 / 7530-14269 JSONTXT

{"project":"LitCovid-PubTator","denotations":[{"id":"175","span":{"begin":12,"end":15},"obj":"Gene"},{"id":"183","span":{"begin":127,"end":130},"obj":"Gene"},{"id":"184","span":{"begin":235,"end":238},"obj":"Gene"},{"id":"185","span":{"begin":529,"end":532},"obj":"Gene"},{"id":"186","span":{"begin":229,"end":234},"obj":"Species"},{"id":"187","span":{"begin":361,"end":368},"obj":"Species"},{"id":"188","span":{"begin":737,"end":744},"obj":"Species"},{"id":"189","span":{"begin":805,"end":808},"obj":"Chemical"},{"id":"195","span":{"begin":1185,"end":1188},"obj":"Gene"},{"id":"196","span":{"begin":1291,"end":1313},"obj":"Chemical"},{"id":"197","span":{"begin":1314,"end":1328},"obj":"Chemical"},{"id":"198","span":{"begin":1350,"end":1353},"obj":"Chemical"},{"id":"199","span":{"begin":1449,"end":1452},"obj":"Chemical"},{"id":"209","span":{"begin":2016,"end":2019},"obj":"Gene"},{"id":"210","span":{"begin":2028,"end":2031},"obj":"Gene"},{"id":"211","span":{"begin":2270,"end":2273},"obj":"Gene"},{"id":"212","span":{"begin":2096,"end":2103},"obj":"Species"},{"id":"213","span":{"begin":1626,"end":1629},"obj":"Chemical"},{"id":"214","span":{"begin":1751,"end":1767},"obj":"Chemical"},{"id":"215","span":{"begin":1920,"end":1936},"obj":"Chemical"},{"id":"216","span":{"begin":2192,"end":2196},"obj":"Chemical"},{"id":"217","span":{"begin":2537,"end":2548},"obj":"Chemical"},{"id":"222","span":{"begin":2943,"end":2946},"obj":"Gene"},{"id":"223","span":{"begin":4066,"end":4069},"obj":"Gene"},{"id":"224","span":{"begin":2808,"end":2815},"obj":"Chemical"},{"id":"225","span":{"begin":4189,"end":4199},"obj":"Chemical"},{"id":"234","span":{"begin":4783,"end":4786},"obj":"Gene"},{"id":"235","span":{"begin":5124,"end":5127},"obj":"Gene"},{"id":"236","span":{"begin":5183,"end":5186},"obj":"Gene"},{"id":"237","span":{"begin":4216,"end":4223},"obj":"Chemical"},{"id":"238","span":{"begin":4332,"end":4339},"obj":"Chemical"},{"id":"239","span":{"begin":4659,"end":4677},"obj":"Chemical"},{"id":"240","span":{"begin":5403,"end":5411},"obj":"Chemical"},{"id":"241","span":{"begin":5043,"end":5054},"obj":"Disease"},{"id":"243","span":{"begin":5631,"end":5634},"obj":"Gene"},{"id":"252","span":{"begin":5959,"end":5962},"obj":"Gene"},{"id":"253","span":{"begin":6112,"end":6115},"obj":"Gene"},{"id":"254","span":{"begin":5804,"end":5808},"obj":"Species"},{"id":"255","span":{"begin":6108,"end":6111},"obj":"Species"},{"id":"256","span":{"begin":6162,"end":6167},"obj":"Species"},{"id":"257","span":{"begin":6561,"end":6565},"obj":"Species"},{"id":"258","span":{"begin":5731,"end":5751},"obj":"Chemical"},{"id":"259","span":{"begin":5849,"end":5856},"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"},{"id":"A209","pred":"tao:has_database_id","subj":"209","obj":"Gene:134864"},{"id":"A210","pred":"tao:has_database_id","subj":"210","obj":"Gene:134864"},{"id":"A211","pred":"tao:has_database_id","subj":"211","obj":"Gene:134864"},{"id":"A212","pred":"tao:has_database_id","subj":"212","obj":"Tax:562"},{"id":"A214","pred":"tao:has_database_id","subj":"214","obj":"MESH:D000645"},{"id":"A215","pred":"tao:has_database_id","subj":"215","obj":"MESH:D000645"},{"id":"A216","pred":"tao:has_database_id","subj":"216","obj":"MESH:D012492"},{"id":"A222","pred":"tao:has_database_id","subj":"222","obj":"Gene:134864"},{"id":"A223","pred":"tao:has_database_id","subj":"223","obj":"Gene:134864"},{"id":"A224","pred":"tao:has_database_id","subj":"224","obj":"MESH:D019343"},{"id":"A225","pred":"tao:has_database_id","subj":"225","obj":"MESH:D008715"},{"id":"A234","pred":"tao:has_database_id","subj":"234","obj":"Gene:134864"},{"id":"A235","pred":"tao:has_database_id","subj":"235","obj":"Gene:134864"},{"id":"A236","pred":"tao:has_database_id","subj":"236","obj":"Gene:134864"},{"id":"A237","pred":"tao:has_database_id","subj":"237","obj":"MESH:D010455"},{"id":"A240","pred":"tao:has_database_id","subj":"240","obj":"MESH:D010455"},{"id":"A241","pred":"tao:has_database_id","subj":"241","obj":"MESH:D001791"},{"id":"A243","pred":"tao:has_database_id","subj":"243","obj":"Gene:50719"},{"id":"A252","pred":"tao:has_database_id","subj":"252","obj":"Gene:50719"},{"id":"A253","pred":"tao:has_database_id","subj":"253","obj":"Gene:50719"},{"id":"A254","pred":"tao:has_database_id","subj":"254","obj":"Tax:10116"},{"id":"A255","pred":"tao:has_database_id","subj":"255","obj":"Tax:10116"},{"id":"A256","pred":"tao:has_database_id","subj":"256","obj":"Tax:9606"},{"id":"A257","pred":"tao:has_database_id","subj":"257","obj":"Tax:10116"}],"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":"2. Results\n\n2.1. 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.\n\n2.2. Purification and In Vitro Characterization of PASylated Tα1\nThe uncharged PAS moiety, which does not alter the isoelectric point of the target peptide, facilitates classical protein precipitation by ammonium sulfate, thus providing an efficient and inexpensive purification step. After adjusting the cleared whole cell extract prepared by mechanical cell lysis to 30% ammonium sulfate saturation, most of the host cell proteins remained in solution while both PAS-Tα1 and PAS-Tα1 were selectively recovered as a precipitate. To remove residual E. coli proteins, the redissolved precipitate was subjected to ion exchange chromatography on a salt-tolerant anion exchange (AEX) resin at pH 8.5. Even though the PASylated Tα1 peptide with a calculated pI of 4.3 [38] for both versions should be negatively charged under these conditions and, hence, is expected to adsorb to the resin, the recombinant fusion proteins were quantitatively found in the flow-through. Possibly, the voluminous PAS polymer partially shields the small peptide from ionic interactions with the chromatography matrix. Nevertheless, this step resulted in efficient depletion both of residual host cell proteins and of bacterial endotoxins.\nThe protein solutions were dialyzed against a citrate buffer at pH 3.0 and subsequently applied to a strong cation exchange (CEX) column, which resulted in a bound fraction for PAS-Tα1, whereas both a flow-through fraction and a bound fraction were observed for Tα1-PAS (Figure 2). Electrospray ionization mass spectrometry (ESI-MS) analysis of Tα1-PAS in the flow-through revealed a molecular mass of 52,734.56 Da (Figure 2a), which exactly matches the calculated mass for the N-terminally acetylated gene product (52,734.56 Da). In this case, the start methionine of Tα1-PAS (followed by a Ser residue) was fully processed, presumably by the bacterial methionine aminopeptidase [39], then followed by N-terminal acetylation via RimJ. In contrast, the column-bound peptide fraction, which was eluted using a salt concentration gradient, showed a molecular mass of 52,692.38 Da (Figure 2b), which corresponds to the calculated mass for the non-acetylated processed polypeptide (52,692.54 Da) accompanied by some minor peaks below 40 kDa, most likely due to residual host cell impurities. Accordingly, this CEX step enabled separation of the desired N-acetylated Tα1-PAS from its non-acetylated precursor as a result of a single charge difference. In comparison, the fully column-bound non-acetylated PAS-Tα1 showed a single molecular mass of 52,789.8 Da (Figure 3) corresponding to the intact peptide, again, lacking the start methionine.\nBoth PASylated peptide preparations had a purity \u003e 96% as indicated by reverse-phase chromatography (Figure 2c and Figure 3c). For Tα1-PAS, we performed a final AEX polishing step, which also allowed concentration of the peptide. At pH 10, the acetylated Tα1-PAS bound to a strong AEX resin and eluted as a homogenous peak in a salt concentration gradient. The endotoxin content of this fraction was very low, with \u003c 0.1 EU/mg, and the final yield was 15 mg acetylated Tα1-PAS per 1 L bacterial culture. In comparison, the final yield of the fully column-bound (non-acetylated) PAS-Tα1 reached 50 mg per 1 L bacterial culture after CEX chromatography and, again, the endotoxin content was below 0.1 EU/mg. Analytical size exclusion chromatography (SEC) of both PASylated peptide versions revealed a single symmetric peak without any signs of aggregation or truncation (Figure 2d and Figure 3d). The N-terminally acetylated Tα1-PAS eluted at 13.5 mL (bed volume: 24 mL), whereas PAS-Tα1 eluted at 13.2 mL, thus indicating apparent molecular sizes of 557 kDa and 665 kDa, respectively. This is more than 10 times (Tα1-PAS) or even 12 times (PAS-Tα1) larger than the true molecular mass of both PASylated peptides (52.7 kDa), which demonstrates the huge expansion of the hydrodynamic molecular volume caused by the random-coil structure of the PAS polymer, in line with previous observations [35].\n\n2.3. PASylation Strongly Prolongs Tα1 Pharmacokinetics in Rats\nTo mimic the clinically approved route of Zadaxin™ administration, the N-acetylated Tα1-PAS was injected subcutaneously into the dorsal area of rats (N = 5). The injected dose of 3.4 mg/kg Tα1-PAS was well tolerated without any drug-related adverse events or significant changes in body weight. The Tα1-PAS plasma levels at various sampling times were analyzed using a quantitative sandwich ELISA developed to detect only Tα1-PAS and no endogenous rat Tα1, which shares 100% sequence identity with the human peptide. The pharmacokinetic (PK) profile of Tα1-PAS (Figure 4) exhibited a typical curve according to the Bateman function [40], with a Cmax of 25.6 ± 4.4 mg/L at tmax = 22.7 ± 1.1 h. Curve fitting with the WinNonlin software revealed a drastically extended terminal half-life of 15.9 ± 0.9 h, which is more than 8-fold longer than the one for the native peptide (τ1/2 = 1.9 h) published for rats [41]. The strong impact of PASylation on the PK profile is also reflected by other parameters such as the large area under the curve (AUC) and slow clearance (CL) (Table 1)."}

{"project":"LitCovid-sentences","denotations":[{"id":"T46","span":{"begin":0,"end":2},"obj":"Sentence"},{"id":"T47","span":{"begin":3,"end":10},"obj":"Sentence"},{"id":"T48","span":{"begin":12,"end":16},"obj":"Sentence"},{"id":"T49","span":{"begin":17,"end":66},"obj":"Sentence"},{"id":"T50","span":{"begin":67,"end":460},"obj":"Sentence"},{"id":"T51","span":{"begin":461,"end":643},"obj":"Sentence"},{"id":"T52","span":{"begin":644,"end":832},"obj":"Sentence"},{"id":"T53","span":{"begin":833,"end":922},"obj":"Sentence"},{"id":"T54","span":{"begin":923,"end":1228},"obj":"Sentence"},{"id":"T55","span":{"begin":1229,"end":1545},"obj":"Sentence"},{"id":"T56","span":{"begin":1547,"end":1551},"obj":"Sentence"},{"id":"T57","span":{"begin":1552,"end":1611},"obj":"Sentence"},{"id":"T58","span":{"begin":1612,"end":1831},"obj":"Sentence"},{"id":"T59","span":{"begin":1832,"end":2076},"obj":"Sentence"},{"id":"T60","span":{"begin":2077,"end":2243},"obj":"Sentence"},{"id":"T61","span":{"begin":2244,"end":2511},"obj":"Sentence"},{"id":"T62","span":{"begin":2512,"end":2640},"obj":"Sentence"},{"id":"T63","span":{"begin":2641,"end":2761},"obj":"Sentence"},{"id":"T64","span":{"begin":2762,"end":3043},"obj":"Sentence"},{"id":"T65","span":{"begin":3044,"end":3292},"obj":"Sentence"},{"id":"T66","span":{"begin":3293,"end":3497},"obj":"Sentence"},{"id":"T67","span":{"begin":3498,"end":3849},"obj":"Sentence"},{"id":"T68","span":{"begin":3850,"end":4008},"obj":"Sentence"},{"id":"T69","span":{"begin":4009,"end":4200},"obj":"Sentence"},{"id":"T70","span":{"begin":4201,"end":4327},"obj":"Sentence"},{"id":"T71","span":{"begin":4328,"end":4430},"obj":"Sentence"},{"id":"T72","span":{"begin":4431,"end":4557},"obj":"Sentence"},{"id":"T73","span":{"begin":4558,"end":4704},"obj":"Sentence"},{"id":"T74","span":{"begin":4705,"end":4906},"obj":"Sentence"},{"id":"T75","span":{"begin":4907,"end":5095},"obj":"Sentence"},{"id":"T76","span":{"begin":5096,"end":5162},"obj":"Sentence"},{"id":"T77","span":{"begin":5163,"end":5284},"obj":"Sentence"},{"id":"T78","span":{"begin":5285,"end":5595},"obj":"Sentence"},{"id":"T79","span":{"begin":5597,"end":5601},"obj":"Sentence"},{"id":"T80","span":{"begin":5602,"end":5659},"obj":"Sentence"},{"id":"T81","span":{"begin":5660,"end":5817},"obj":"Sentence"},{"id":"T82","span":{"begin":5818,"end":5954},"obj":"Sentence"},{"id":"T83","span":{"begin":5955,"end":6176},"obj":"Sentence"},{"id":"T84","span":{"begin":6177,"end":6352},"obj":"Sentence"},{"id":"T85","span":{"begin":6353,"end":6571},"obj":"Sentence"},{"id":"T86","span":{"begin":6572,"end":6739},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"2. Results\n\n2.1. 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.\n\n2.2. Purification and In Vitro Characterization of PASylated Tα1\nThe uncharged PAS moiety, which does not alter the isoelectric point of the target peptide, facilitates classical protein precipitation by ammonium sulfate, thus providing an efficient and inexpensive purification step. After adjusting the cleared whole cell extract prepared by mechanical cell lysis to 30% ammonium sulfate saturation, most of the host cell proteins remained in solution while both PAS-Tα1 and PAS-Tα1 were selectively recovered as a precipitate. To remove residual E. coli proteins, the redissolved precipitate was subjected to ion exchange chromatography on a salt-tolerant anion exchange (AEX) resin at pH 8.5. Even though the PASylated Tα1 peptide with a calculated pI of 4.3 [38] for both versions should be negatively charged under these conditions and, hence, is expected to adsorb to the resin, the recombinant fusion proteins were quantitatively found in the flow-through. Possibly, the voluminous PAS polymer partially shields the small peptide from ionic interactions with the chromatography matrix. Nevertheless, this step resulted in efficient depletion both of residual host cell proteins and of bacterial endotoxins.\nThe protein solutions were dialyzed against a citrate buffer at pH 3.0 and subsequently applied to a strong cation exchange (CEX) column, which resulted in a bound fraction for PAS-Tα1, whereas both a flow-through fraction and a bound fraction were observed for Tα1-PAS (Figure 2). Electrospray ionization mass spectrometry (ESI-MS) analysis of Tα1-PAS in the flow-through revealed a molecular mass of 52,734.56 Da (Figure 2a), which exactly matches the calculated mass for the N-terminally acetylated gene product (52,734.56 Da). In this case, the start methionine of Tα1-PAS (followed by a Ser residue) was fully processed, presumably by the bacterial methionine aminopeptidase [39], then followed by N-terminal acetylation via RimJ. In contrast, the column-bound peptide fraction, which was eluted using a salt concentration gradient, showed a molecular mass of 52,692.38 Da (Figure 2b), which corresponds to the calculated mass for the non-acetylated processed polypeptide (52,692.54 Da) accompanied by some minor peaks below 40 kDa, most likely due to residual host cell impurities. Accordingly, this CEX step enabled separation of the desired N-acetylated Tα1-PAS from its non-acetylated precursor as a result of a single charge difference. In comparison, the fully column-bound non-acetylated PAS-Tα1 showed a single molecular mass of 52,789.8 Da (Figure 3) corresponding to the intact peptide, again, lacking the start methionine.\nBoth PASylated peptide preparations had a purity \u003e 96% as indicated by reverse-phase chromatography (Figure 2c and Figure 3c). For Tα1-PAS, we performed a final AEX polishing step, which also allowed concentration of the peptide. At pH 10, the acetylated Tα1-PAS bound to a strong AEX resin and eluted as a homogenous peak in a salt concentration gradient. The endotoxin content of this fraction was very low, with \u003c 0.1 EU/mg, and the final yield was 15 mg acetylated Tα1-PAS per 1 L bacterial culture. In comparison, the final yield of the fully column-bound (non-acetylated) PAS-Tα1 reached 50 mg per 1 L bacterial culture after CEX chromatography and, again, the endotoxin content was below 0.1 EU/mg. Analytical size exclusion chromatography (SEC) of both PASylated peptide versions revealed a single symmetric peak without any signs of aggregation or truncation (Figure 2d and Figure 3d). The N-terminally acetylated Tα1-PAS eluted at 13.5 mL (bed volume: 24 mL), whereas PAS-Tα1 eluted at 13.2 mL, thus indicating apparent molecular sizes of 557 kDa and 665 kDa, respectively. This is more than 10 times (Tα1-PAS) or even 12 times (PAS-Tα1) larger than the true molecular mass of both PASylated peptides (52.7 kDa), which demonstrates the huge expansion of the hydrodynamic molecular volume caused by the random-coil structure of the PAS polymer, in line with previous observations [35].\n\n2.3. PASylation Strongly Prolongs Tα1 Pharmacokinetics in Rats\nTo mimic the clinically approved route of Zadaxin™ administration, the N-acetylated Tα1-PAS was injected subcutaneously into the dorsal area of rats (N = 5). The injected dose of 3.4 mg/kg Tα1-PAS was well tolerated without any drug-related adverse events or significant changes in body weight. The Tα1-PAS plasma levels at various sampling times were analyzed using a quantitative sandwich ELISA developed to detect only Tα1-PAS and no endogenous rat Tα1, which shares 100% sequence identity with the human peptide. The pharmacokinetic (PK) profile of Tα1-PAS (Figure 4) exhibited a typical curve according to the Bateman function [40], with a Cmax of 25.6 ± 4.4 mg/L at tmax = 22.7 ± 1.1 h. Curve fitting with the WinNonlin software revealed a drastically extended terminal half-life of 15.9 ± 0.9 h, which is more than 8-fold longer than the one for the native peptide (τ1/2 = 1.9 h) published for rats [41]. The strong impact of PASylation on the PK profile is also reflected by other parameters such as the large area under the curve (AUC) and slow clearance (CL) (Table 1)."}