Size exclusion column

The preparation method of A3A(E72A/C171A) protein was described previously26 (link) as follows: the protein was expressed in E. coli strain BL21 DE3 Star (Stratagene) cells with pCold-GST-A3A(E72A/C171A) vector. Expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 16 °C for 22 h in lysogeny broth medium containing 100 μg ml^−l ampicillin. Cells were pelleted, resuspended in purification buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 1 mM dithiothreitol) and lysed through sonication. Cellular debris was separated by centrifugation (45,000g, 30 min, 4 °C). The protein was purified as a GST-fused protein with glutathione-immobilized resin (Clontech). After digesting with HRV 3C protease, the protein was further purified with a size-exclusion column (GE Healthcare) equilibrated with a buffer (10 mM Tris-HCl (pH 8.0), 200 mM NaCl and 1 mM dithiothreitol). The fraction containing the monomeric form was collected and concentrated for crystallization. The purity and integrity of A3A(E72A/C171A) was confirmed by SDS–polyacrylamide gel electrophoresis. E72A inactivates the enzyme, while C171A (distal the active site) enhances solubility of the expressed protein.
The DNA oligo, d(TTTTTTTTCTTTTTT), was synthesized (Integrated DNA Technologies), and mixed with the purified A3A(E72A/C171A) protein at a molar ratio of 2:1.

Wild-type PaFS was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL (Novagen). Cells were grown at 37°C with aeration following inoculation of 6 × 1 L 2xYT medium containing 50 µg/mL kanamycin and 30 µg/mL chloramphenicol with 5 mL of starting culture. Once the optical density at 600 nm reached 0.8, cells were induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside at 16°C followed by 18 h expression. Cells were pelleted by centrifugation and resuspended in wash buffer [50 mM K₂HPO₄ (pH 7.5), 300 mM NaCl, 10% (v/v) glycerol, 1.5 mM tris(2-carboxyethyl)-phosphine hydrochloride (TCEP)]. After lysis by sonication, cell lysate was clarified by centrifugation and loaded on a Ni-NTA column (GE Healthcare). Protein was eluted with a 0–400 mM imidazole gradient in wash buffer. Fractions were analyzed by SDS-PAGE, and were combined and loaded onto a size-exclusion column (GE Healthcare) pre-equilibrated with 50 mM Tris (pH 7.7), 200 mM NaCl, 10% (v/v) glycerol, and 1.5 mM TCEP. The protein sample was ~99% pure as judged by SDS-PAGE. Prior to the preparation of grids for EM and cryo-EM, PaFS was dialyzed into EM buffer [2.0 mg/mL PaFS, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5), 150 mM NaCl, 1.5 mM TCEP].

Antibodies were produced by transient co-expression of Expi293 cells with pcDNA3.4-Heavy and pcDNA3.4-Light expression vectors (Life Technologies, Carlsbad, USA) in serum-free expression medium (Life Technologies) according to the supplier’s recommended protocol. For co-transfections, plasmids were transfected in the same mass ratio into mammalian cells which were supplemented with enhancer the next day. Cell culture supernatants were harvested 5 days thereafter. SDS-PAGE was used to detect the protein bands. Antibodies were purified by protein A column (GE Healthcare, Pittsburgh, USA) and size exclusion column (GE Healthcare) following buffer exchange in PBS (pH 7.2). Antibody concentration was measured by NanoDrop at 280 nm. The protein purity was analyzed by SDS-PAGE and SEC-HPLC.

N300 was expressed in E. coli LOBSTER strain and was purified using a Ni²⁺-chelating affinity column. 6 × His-tag was removed by TEV cleavage overnight at 4°C. The N300 was further purified by FPLC using a heparin column and a size exclusion column (GE Healthcare). The final storage buffer for N300 contained 20 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, 1 mM magnesium chloride, and 5 mM β-mercaptoethanol (BME).
The selenomethionine (SeMet) substituted N300 sample was expressed using a previously described method (Van Duyne et al., 1993 (link)) and then purified using the same procedure. The final storage buffer for N300 contained 20 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, and 5 mM BME.