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PMC:7795856 / 7530-14269 JSONTXT

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LitCovid-PubTator

Id Subject Object Predicate Lexical cue tao:has_database_id
175 12-15 Gene denotes 2.1 Gene:6700
183 127-130 Gene denotes Tα1 Gene:134864
184 235-238 Gene denotes Tα1 Gene:134864
185 529-532 Gene denotes Tα1 Gene:134864
186 229-234 Species denotes human Tax:9606
187 361-368 Species denotes E. coli Tax:562
188 737-744 Species denotes E. coli Tax:562
189 805-808 Chemical denotes tet MESH:C010349
195 1185-1188 Gene denotes Tα1 Gene:134864
196 1291-1313 Chemical denotes sodium dodecyl sulfate MESH:D012967
197 1314-1328 Chemical denotes polyacrylamide MESH:C016679
198 1350-1353 Chemical denotes SDS MESH:D012967
199 1449-1452 Chemical denotes SDS MESH:D012967
209 2016-2019 Gene denotes Tα1 Gene:134864
210 2028-2031 Gene denotes Tα1 Gene:134864
211 2270-2273 Gene denotes Tα1 Gene:134864
212 2096-2103 Species denotes E. coli Tax:562
213 1626-1629 Chemical denotes PAS
214 1751-1767 Chemical denotes ammonium sulfate MESH:D000645
215 1920-1936 Chemical denotes ammonium sulfate MESH:D000645
216 2192-2196 Chemical denotes salt MESH:D012492
217 2537-2548 Chemical denotes PAS polymer
222 2943-2946 Gene denotes Tα1 Gene:134864
223 4066-4069 Gene denotes Tα1 Gene:134864
224 2808-2815 Chemical denotes citrate MESH:D019343
225 4189-4199 Chemical denotes methionine MESH:D008715
234 4783-4786 Gene denotes Tα1 Gene:134864
235 5124-5127 Gene denotes Tα1 Gene:134864
236 5183-5186 Gene denotes Tα1 Gene:134864
237 4216-4223 Chemical denotes peptide MESH:D010455
238 4332-4339 Chemical denotes Tα1-PAS
239 4659-4677 Chemical denotes acetylated Tα1-PAS
240 5403-5411 Chemical denotes peptides MESH:D010455
241 5043-5054 Disease denotes aggregation MESH:D001791
243 5631-5634 Gene denotes Tα1 Gene:50719
252 5959-5962 Gene denotes Tα1 Gene:50719
253 6112-6115 Gene denotes Tα1 Gene:50719
254 5804-5808 Species denotes rats Tax:10116
255 6108-6111 Species denotes rat Tax:10116
256 6162-6167 Species denotes human Tax:9606
257 6561-6565 Species denotes rats Tax:10116
258 5731-5751 Chemical denotes N-acetylated Tα1-PAS
259 5849-5856 Chemical denotes Tα1-PAS

LitCovid-sentences

Id Subject Object Predicate Lexical cue
T46 0-2 Sentence denotes 2.
T47 3-10 Sentence denotes Results
T48 12-16 Sentence denotes 2.1.
T49 17-66 Sentence denotes Cloning and Bacterial Production of PASylated Tα1
T50 67-460 Sentence denotes To 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].
T51 461-643 Sentence denotes 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).
T52 644-832 Sentence denotes 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].
T53 833-922 Sentence denotes The whole cell lysate was analyzed prior to and 15 h after induction by Western blotting.
T54 923-1228 Sentence denotes 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.
T55 1229-1545 Sentence denotes 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.
T56 1547-1551 Sentence denotes 2.2.
T57 1552-1611 Sentence denotes Purification and In Vitro Characterization of PASylated Tα1
T58 1612-1831 Sentence denotes The 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.
T59 1832-2076 Sentence denotes 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.
T60 2077-2243 Sentence denotes 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.
T61 2244-2511 Sentence denotes 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.
T62 2512-2640 Sentence denotes Possibly, the voluminous PAS polymer partially shields the small peptide from ionic interactions with the chromatography matrix.
T63 2641-2761 Sentence denotes Nevertheless, this step resulted in efficient depletion both of residual host cell proteins and of bacterial endotoxins.
T64 2762-3043 Sentence denotes The 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).
T65 3044-3292 Sentence denotes 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).
T66 3293-3497 Sentence denotes 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.
T67 3498-3849 Sentence denotes 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.
T68 3850-4008 Sentence denotes 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.
T69 4009-4200 Sentence denotes 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.
T70 4201-4327 Sentence denotes Both PASylated peptide preparations had a purity > 96% as indicated by reverse-phase chromatography (Figure 2c and Figure 3c).
T71 4328-4430 Sentence denotes For Tα1-PAS, we performed a final AEX polishing step, which also allowed concentration of the peptide.
T72 4431-4557 Sentence denotes At pH 10, the acetylated Tα1-PAS bound to a strong AEX resin and eluted as a homogenous peak in a salt concentration gradient.
T73 4558-4704 Sentence denotes The endotoxin content of this fraction was very low, with < 0.1 EU/mg, and the final yield was 15 mg acetylated Tα1-PAS per 1 L bacterial culture.
T74 4705-4906 Sentence denotes 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.
T75 4907-5095 Sentence denotes 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).
T76 5096-5162 Sentence denotes The N-terminally acetylated Tα1-PAS eluted at 13.5 mL (bed volume:
T77 5163-5284 Sentence denotes 24 mL), whereas PAS-Tα1 eluted at 13.2 mL, thus indicating apparent molecular sizes of 557 kDa and 665 kDa, respectively.
T78 5285-5595 Sentence denotes 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].
T79 5597-5601 Sentence denotes 2.3.
T80 5602-5659 Sentence denotes PASylation Strongly Prolongs Tα1 Pharmacokinetics in Rats
T81 5660-5817 Sentence denotes To mimic the clinically approved route of Zadaxin™ administration, the N-acetylated Tα1-PAS was injected subcutaneously into the dorsal area of rats (N = 5).
T82 5818-5954 Sentence denotes 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.
T83 5955-6176 Sentence denotes 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.
T84 6177-6352 Sentence denotes 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.
T85 6353-6571 Sentence denotes 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].
T86 6572-6739 Sentence denotes 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).