> top > docs > PMC:7795856 > spans > 9077-13125 > annotations

PMC:7795856 / 9077-13125 JSONTXT

Annnotations TAB JSON ListView MergeView

LitCovid-PubTator

Id Subject Object Predicate Lexical cue tao:has_database_id
209 469-472 Gene denotes Tα1 Gene:134864
210 481-484 Gene denotes Tα1 Gene:134864
211 723-726 Gene denotes Tα1 Gene:134864
212 549-556 Species denotes E. coli Tax:562
213 79-82 Chemical denotes PAS
214 204-220 Chemical denotes ammonium sulfate MESH:D000645
215 373-389 Chemical denotes ammonium sulfate MESH:D000645
216 645-649 Chemical denotes salt MESH:D012492
217 990-1001 Chemical denotes PAS polymer
222 1396-1399 Gene denotes Tα1 Gene:134864
223 2519-2522 Gene denotes Tα1 Gene:134864
224 1261-1268 Chemical denotes citrate MESH:D019343
225 2642-2652 Chemical denotes methionine MESH:D008715
234 3236-3239 Gene denotes Tα1 Gene:134864
235 3577-3580 Gene denotes Tα1 Gene:134864
236 3636-3639 Gene denotes Tα1 Gene:134864
237 2669-2676 Chemical denotes peptide MESH:D010455
238 2785-2792 Chemical denotes Tα1-PAS
239 3112-3130 Chemical denotes acetylated Tα1-PAS
240 3856-3864 Chemical denotes peptides MESH:D010455
241 3496-3507 Disease denotes aggregation MESH:D001791

LitCovid-sentences

Id Subject Object Predicate Lexical cue
T56 0-4 Sentence denotes 2.2.
T57 5-64 Sentence denotes Purification and In Vitro Characterization of PASylated Tα1
T58 65-284 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 285-529 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 530-696 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 697-964 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 965-1093 Sentence denotes Possibly, the voluminous PAS polymer partially shields the small peptide from ionic interactions with the chromatography matrix.
T63 1094-1214 Sentence denotes Nevertheless, this step resulted in efficient depletion both of residual host cell proteins and of bacterial endotoxins.
T64 1215-1496 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 1497-1745 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 1746-1950 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 1951-2302 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 2303-2461 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 2462-2653 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 2654-2780 Sentence denotes Both PASylated peptide preparations had a purity > 96% as indicated by reverse-phase chromatography (Figure 2c and Figure 3c).
T71 2781-2883 Sentence denotes For Tα1-PAS, we performed a final AEX polishing step, which also allowed concentration of the peptide.
T72 2884-3010 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 3011-3157 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 3158-3359 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 3360-3548 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 3549-3615 Sentence denotes The N-terminally acetylated Tα1-PAS eluted at 13.5 mL (bed volume:
T77 3616-3737 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 3738-4048 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].