Akta explorer

The expression and purification of recombinant TTR (possessing or devoid of the C-terminal histidine tag), which was cloned into the pGEX-2T vector, was performed according to Ref. [34 (link)]. Briefly, the expression of TTR fused to glutathione-S-transferase (GST) in the E. coli BL21 pLys cell line was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside. The cells were cultured for 3 h at 37 °C, harvested by centrifugation, washed and suspended in phosphate-buffered saline (PBS) containing 1 mM DTT and frozen at −80 °C. Prior to purification, the cell suspension was thawed and lysed by pipetting. DNase I (10 μg/mL), RNase A (10 μg/mL) and phenylmethylsulfonyl fluoride (0.2 mg/mL) were added during cell lysis, and the soluble fraction was obtained by centrifugation. TTR was purified using a two-step protocol. First, the supernatant (containing fusion protein) was loaded on a glutathione-Sepharose column equilibrated with PBS, and contaminating proteins were washed out with PBS. Then, TTR was digested from the resin with thrombin (Calbiochem) at room temperature for up to 24 h. TTR-containing fractions were collected by washing the column with PBS, concentrated using a Centricon filter unit (Millipore), loaded onto a Superdex 200 column (30/300 GL, GE Healthcare) equilibrated with Tris buffer and separated using Akta Explorer (Amersham Biosciences).

Overnight 5 mL liquid cultures of yeast strains in synthetic defined dropout media were diluted into 50 mL of fresh media and grown at 30 ^oC for 18–24 h. Cells were resuspended in 6 mL PBS buffer and lysed using glass beads, and then sodium chloride and PEG-8000 were added to the soluble fraction to a final concentration of 0.5 M and 8%, respectively. After sitting for 15 min on ice, the precipitate was isolated, redissolved in 2 mL PBS buffer, and purified by size exclusion using a HiPrep 16/60 Sephacryl S-500 HR column (GE Healthcare) in PBS buffer (1 mL/min) on an AKTA Explorer (Amersham Biosciences). The encapsulin fractions were concentrated using Amicon Ultra-15 Centrifugal Filter Units with Ultracel-100 membrane (Millipore), then diluted in 2 mL of 20 mM Tris buffer at pH 8. Ion-exchange chromatography using a HiPrep DEAE FF 16/10 column (GE Healthcare) resulted in the fully purified encapsulin sample for further analysis. The gradient used for ion-exchange was as follows: 100% A for 0–100 mL, 100% A to 50% A + 50% B for 100–200 mL, 100% B for 200–300 mL, 100% A for 300–400 mL; where A is 20 mM Tris pH 8, B is 20 mM Tris pH 8 with 1 M NaCl (flow rate: 3 mL/min). Examples of purification chromatographs can be found in Supplementary Figs. 13 and 14.

Recombinant TTR (rTTR) fused with glutathione-S-transferase was expressed in the E. coli BL21 pLys cell line. After inducing protein expression with 1-mM isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were cultured for 3 h at 37°C, harvested by centrifugation, washed and suspended in phosphate-buffered saline (PBS) containing 1-mM dithiotreitol (DTT) and frozen at -80°C. Prior to purification, the cell suspension was thawed, and the cells were lysed by pipetting. DNase I, RNAse A and phenylmethylsulfonyl fluoride were added to final concentrations of 10 μg/ml, 10 μg/ml and 0.2 μg/ml, respectively. The soluble fraction was obtained by centrifugation for at least 1 h at 18000xg and was loaded on a glutathione-sepharose column equilibrated with PBS. Contaminating proteins were washed out with PBS, and rTTR was digested out of the glutathione-S-transferase with thrombin (Calbiochem) at room temperature for up to 24 h. rTTR-containing fractions were collected by washing the column with PBS, concentrated using a centricon (Millipore), loaded onto a Superdex 200 column (GE Healthcare, 17-5175-01) and separated using Akta Explorer (Amersham Biosciences).