| Id |
Subject |
Object |
Predicate |
Lexical cue |
| T48 |
0-4 |
Sentence |
denotes |
2.1. |
| T49 |
5-54 |
Sentence |
denotes |
Cloning and Bacterial Production of PASylated Tα1 |
| T50 |
55-448 |
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 |
449-631 |
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 |
632-820 |
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 |
821-910 |
Sentence |
denotes |
The whole cell lysate was analyzed prior to and 15 h after induction by Western blotting. |
| T54 |
911-1216 |
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 |
1217-1533 |
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. |
