| Id |
Subject |
Object |
Predicate |
Lexical cue |
| T219 |
0-45 |
Sentence |
denotes |
Cloning, Protein Expression, and Purification |
| T220 |
46-299 |
Sentence |
denotes |
The MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence. |
| T221 |
300-464 |
Sentence |
denotes |
Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3. |
| T222 |
465-539 |
Sentence |
denotes |
The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells. |
| T223 |
540-749 |
Sentence |
denotes |
The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h. |
| T224 |
750-917 |
Sentence |
denotes |
The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5). |
| T225 |
918-1009 |
Sentence |
denotes |
The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris. |
| T226 |
1010-1178 |
Sentence |
denotes |
The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM. |
| T227 |
1179-1347 |
Sentence |
denotes |
Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada). |
