Arctic express de3 ril

To create WRC:Abi2-(MBP)₂, the sortase ligation sequence, LPGTG, was genetically fused to the C-terminus of MBP-Abi2 (1-158). Meanwhile, a TEV site was added to the N-terminus of an (MBP)₂ tag, which after Tev cleavage would expose a Gly required for sortase ligation. MBP-Abi2 (1-158)-LPGTG was expressed, purified, and incorporated into the WRC as described above to create WRC-LPGTG. GG-2MBP was expressed in Arctic Express (DE3) RIL (Agilent) cells after induction with 0.75 mM IPTG at 10°C for 16 hr, purified on amylose resin, and subjected to TEV cleavage overnight, followed by anion exchange chromatography using a Source 15Q column (GE Healthcare). Sortase5M (sortase A pentamutant) was a gift from David Liu (Addgene plasmid # 75144), expressed in Arctic Express (DE3) RIL (Agilent) cells, purified over Ni-NTA agarose resin, followed by cation exchange using a Source 15S column (GE Healthcare) and size exclusion chromatography using a Superdex 75 column (GE Healthcare) (Chen et al., 2011 (link)). WRC-LPGTG (1 µM) was mixed with GG-MBP (25 µM) and sortase (10 µM) in 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM CaCl₂ and left at RT for 2 hr. The reaction was quenched by adding 25 mM EGTA, and the WRC-(MBP)₂ was purified over a Superdex 200 column to separate the WRC-(MBP)₂ from unligated molecules and sortase.

Escherichia coli ArcticExpress (DE3) RIL (Agilent Technologies) transfected with pET21b(+)/KnRAV-DBD was pre-cultured in 20 mL of LB medium containing 100 mg/L ampicillin, 20 mg/L gentamicin, and 1% glucose at 37 °C overnight with shaking. Then, 10 mL of the overnight culture was each inoculated into two 500 mL volumes of LB medium without antibiotics and cultured at 30 °C with shaking. After growing to OD₆₀₀ = 0.5, the culture medium was transferred to 13 °C and incubated overnight in the presence of 0.4 mM isopropyl-β-d-thiogalactopyranoside. The cells were collected by centrifugation at 2000×g at 4 °C for 10 min and suspended in 12.5 mL sonication buffer (20 mM Tris-HCl pH 8.0; 300 mM NaCl; 1 mM dithiothreitol), and the cell suspension was disrupted with an ULTRASONIC DISRUPTOR UD-211 (TOMY) and centrifuged at 20,000×g at 4 °C for 5 min. The supernatant was collected in another tube and used for His-tag purification with His60 Ni Superflow (Clontech) and a TALON 2 mL Disposable Gravity Column (TaKaRa) in accordance with the instruction manual. To remove the imidazole-containing elution buffer, the column eluent was dialyzed in 500 mL storage buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). During dialysis overnight at 4 °C, the buffer was replaced twice. The dialyzed content was divided into small aliquots and stored at − 80 °C until use.