Hiload 16 600 superdex 200 pg

GephE (gephyrin P2 splice variant residues 318–736) was expressed in Escherichia coli and purified in a two-step purification as described earlier^{29 (link), 44 (link)}. Concisely, the protein was purified using via Intein-tag (Chitin beads, New England BioLabs), and after self-cleavage the protein could be obtained by size-exclusion chromatography (SEC) column (HiLoad 16/600 Superdex 200 pg, GE Healthcare) on an ÄKTA explorer system (GE Healthcare). His-tagged gephE was produced similarly with the exception of purification on IMAC coloumns before SEC purification.

The crosslinking reactions were carried out by mixing crypto-AcpP with its partner protein (FabF or FabB) in 5:1 ratio at 37 °C for 16 h. Reactions were examined by 12% SDS PAGE and purified by HiLoad 16/600 Superdex 200 PG (GE Biosciences) size exclusion column using minimal buffer (20 mM Tris, 50 mM NaCl, pH 7.4). The resulting protein complex was greater than 95% in purity, determined by SDS PAGE, and was concentrated to 8–10 mg·mL^–1 using Amicon Ultra Centrifuge Filters (MilliporeSigma) with 30 kDa molecular weight cut off. The concentrated crosslinked complex was immediately used for protein crystallization or flash-frozen and stored in −80 °C freezer for later use.

scFvconstructs were cloned into the pTT3 plasmid vector. ScFvs contain only the Fv antibody domains of VH/VL connected by an amino acid linker GGGGSGGGGSGGGGS from the C-terminus of the light chain to the N-terminus of the heavy chain. Iv8 scFv and glVRC01 scFv were expressed using HEK293ENBA cells (Tom et al., 2008 (link)). Cells were cultured in suspension and transfected with 500 µg/liter plasmid DNA using 293 Free Transfection Reagent (Novagen). After 6 d, cells were centrifuged at 4,500 rpm for 20 min. Supernatant was filter sterilized and incubated with His60 Ni-Superflow Resin (Novagen) overnight at 4°C, capturing scFv’s with a 6his-tag on the C-terminus of the proteins. The Ni resin was separated from the supernatant and washed with a solution of 150 mM NaCl, 20 mM Tris (pH 8.0), 20 mM imidazole (pH 7.0) and eluted with a solution of 300 mM NaCl, 50 mM Tris pH 8.0, 250 mM imidazole (pH 7.0). To separate the scFv monomers from diabodies and triabodies and to further purify the proteins, the sample was run on size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg (GE) column. Complexes of iv8 scFv and glVRC01 scFv were formed by mixing proteins at a 1:1 molar ratio. ScFv complexes were removed from monomeric scFv using size-exclusion chromatography. Complexes were concentrated to ∼10 mg/ml for crystallization trials.

The DNA fragment of ASFV B646L gene (GenBank: QBH90570.1) was codon-optimized and cloned into pCAGGS-His vector. The plasmids expressing p72 was transfected to CHO cells using TransIntro^® EL/PL Transfection Reagent (No FT231-02, TrasnGen Biotech, Beijing, China). After 3–4 days post transfection, the cells were harvested and resuspended in phosphate-buffered saline (PBS, pH 7.5) containing protease inhibitor cocktail (No539133, Solarbio, Beijing, China). The soluble p72 was purified by BeaverBeads™ IDA-Nickel Kit-10 (No70501-K10, Beaver, Suzhou, China). The purified sample was subjected to gel filtration chromatography with HiLoad^® 16/600 Superdex^® 200 pg (No 28989335, GE Healthcare, Uppsala, Sweden). The eluted sample was analyzed by in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting (IB) using ASFV-positive standard serum. The soluble p72 was dissolved in PBS (pH 7.5), at a concentration of 1 mg/ml, and coupled to HRP using a Lightning-Link HRP conjugation kit (No701-0010; Innova Biosciences, Cambridge, United Kingdom) according to the instructions.

Monomer RBD protein of SARS-CoV-2 was expressed and purified as previously described [11 (link)]. Briefly, signal peptide sequence of MERS-CoV S protein (MIHSVFLLMFLLTPTES) was added to the RBD protein (S protein 319-541, GenBank: YP_009724390) N terminus for protein secretion, and a hexa-His tag was added to the C terminus to facilitate further purification processes. The coding sequence was codon-optimized for mammalian cell expression and synthesized by GENEWIZ, China. Then, the construct was cloned into the pCAGGS vector and transiently transfected into HEK 293T cells. After 3 days, the supernatant was collected and soluble protein was purified by Ni affinity chromatography using a HisTrap™ HP 5 mL column (GE Healthcare). The sample was further purified via gel filtration chromatography with HiLoad^® 16/600 Superdex^® 200 pg (GE Healthcare) in a buffer composed of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl.

DNA constructs of tandem immunoglobulin domains FLN5 and FLN6, and of poly(GS) glycine serine repeat sequence variants and Y719E variants, were described previously^{20 (link)}. Aromatic and glutamate mutants were generated using site-directed mutagenesis. A stronger SecM variant was used for preparation of [²H,¹³CH₃-Ile, Leu and Val (ILV)]-labelled RNCs for PEGylation experiments, with sequence ‘FSTPVWIWWWPRIRGPPPPWT’. ¹⁵N and [²H,¹³CH₃-ILV]-labelled proteins were grown and purified as described previously^{49 (link)}, except in the case of disordered FLN5 variants where the final chromatography step was performed using a HiLoad 16/600 Superdex 200 pg (GE Healthcare) column in the presence of 6 M urea, followed by buffer exchange into Tico buffer^{25 (link)}. ¹⁵N and [²H,¹³CH₃-ILV]-labelled RNCs were generated in BL21(DE3) Escherichia coli as described previously^{25 (link)}, where the only modification was the replacement of the final step of purification (sucrose gradient chromatography) with an additional sucrose cushion step followed by hydrophobic affinity chromatography using a HiTrap FF butyl column (GE Healthcare). The occupancy and integrity of RNC samples were monitored as described previously^{25 (link)}.

For native protein production, the corresponding pET28a construct was transformed into the E. coli BL21 (DE3) and protein expression was induced in terrific broth media at 25 °C for 16 h after OD₆₀₀ reached 0.6∼0.8. The E. coli cells were harvested by centrifugation at 5,000 g for 15 min and re-suspended in buffer (20 mM HEPES, 150 mM NaCl, 10 mM imidazole pH 7.5). The cells were lysed by sonication on ice. Subsequently, N-His₆-KDM4A/B-DTD was purified via HisTrap HP column (GE Healthcare) with linear elution gradient of imidazole (10–500 mM). The His₆-tag of the proteins was cleaved by thrombin (Sigma) at 4 °C overnight in buffer (20 mM HEPES, 150 mM NaCl pH 7.5, 0.5 mM TCEP). Gel filtration (HiLoad 16/600 Superdex 200 pg, GE Healthcare) was performed in buffer (20 mM HEPES, 150 mM NaCl pH 7.5, 0.5 mM TCEP) to further purify cleaved proteins. The KDM4A-DTD and KDM4B-DTD were concentrated using Amicon Ultra-15 (3000 NMWL, Millipore) to 45 and 38 mg ml⁻¹, respectively, flash frozen in liquid nitrogen and stored at −80 °C.