Complete his tag purification resin

Transfected with either pcDNA3.1-NS3-HIS or RG204546-GEF-H1-GFP or co-transfected with both plasmids, 293T cells were lysed in a NP40 buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40). Cell debris was discarded, and lysates were incubated with cOmplete^TM His-Tag Purification Resin (Roche, Basel, Switzerland) for 1 h at RT or at 4 °C overnight. The Lysate and resin mixes were processed according to the manufacturer’s protocol, resulting in fractionation of His resin-bound proteins and residual cellular proteins.

The expression and purification of the PDI-b’a’ (residues 208–449) and PDI-b’a’ ^C365S/C368S from Humicola insolens were performed as previously described [8 (link),11 (link)]. YFP-PDI-b’a’-CFP and their variants were expressed with an N-terminal hexahistidine tag using the pCold-I vector in E. coli and purified using a Ni²⁺-immobilized affinity column (cOmplete^TM His-Tag Purification Resin, Roche) from the soluble lysate. After the cleavage of the hexahistidine tag with TEV protease, these proteins were purified using a HiLoad Superdex 200 column (Cytiva) in 50 mM Tris–HCl (pH 8.0).
To oxidize the a’ active site, the purified protein (1 mg/mL) was dialyzed using 50 mM Tris-HCl (pH 8.0) containing 0.1 mM oxidized glutathione for 10 days. To reduce the a’ active site, the protein was dissolved in a buffer containing 2–4 mM DTT (Sigma Aldrich, St. Louis, MO, USA).

The conditioned media from HEK293 cells that stably expressed GAL3BP with Myc/His tags were subjected to cOmplete^TM His tag purification resin (Roche, Basel, Switzerland), and bound materials were eluted with TBS containing 0.5 m imidazole. The solvent of the elution fraction was replaced by PBS and concentrated using Amicon Ultra 30-kDa centrifugal filter units (Millipore). The concentrated fraction was used as a recombinant GAL3BP. The preparations obtained in the same processes from mock-transfected HEK293 cells were used as negative controls (vehicle). We also used a commercially available GAL3BP purified from a mouse myeloma cell line, the NS0-derived human galectin-3BP/MAC-2BP protein Val¹⁹–Ala⁵⁸⁵ with a C-terminal His₁₀ tag (R&D Systems).

The 293T/17 cells secreting BiKE or sdCD16 were expanded at 37 °C, and then transferred to a 32 °C incubator for 2–3 days in the presence of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) to maximize protein production. Supernatants from the 293T/17 cells containing recombinant proteins were collected and subsequently purified using cOmplete^TM His-Tag Purification Resin (Roche, Basel, Switzerland). The resin was incubated with supernatants overnight at 4 °C on a rotating shaker. The resin was transferred in cartridges and washed with at least 20 column volumes of buffer A (50 mM Tris-HCl, 1 M NaCl, pH 8.0). Protein was eluted with buffer B (50 mM Tris-HCl, 1 M NaCl, 250 mM imidazole, pH 8.0). Eluted fractions were monitored using a Bradford Coomassie Blue Reagent (Cat 1856209, Lot WA314734; Thermo Scientific, Rockford, IL, USA). Fractions with the greatest signal were pooled and dialyzed using 1× PBS overnight at 4 °C. Protein was then concentrated using an Amicon Ultra-0.5 Centrifugal Filter Unit (10 kDa cutoff) (Millipore Sigma, Burlington, MA, USA). The protein concentration was measured using the NanoDrop (Thermo Scientific) and stored at −80 °C until use.

Recombinant Polydom, Polydom fragments, FLAG-tagged Fc-fusion proteins, and FLAG-tagged COMP-Ang1 were produced using a Freestyle 293 Expression System (Thermo Fisher Scientific). Freestyle 293 cells were transfected with expression vectors using 293fectin (Thermo Fisher Scientific) and grown in serum-free FreeStyle 293 expression medium for 72 h. The conditioned media were collected and clarified by centrifugation. For purification of FLAG-tagged proteins, conditioned media were applied to a DDDDK-tagged protein purification gel (MBL) column and bound proteins were eluted with 100 μg/ml DDDDK-tagged peptide in TBS. For purification of His-tagged proteins, conditioned media were subjected to affinity chromatography using cOmplete His-tag purification resin (Roche). The columns were washed with TBS containing 5 mM imidazole and the bound proteins were eluted with TBS containing 250 mM imidazole. The eluted proteins were dialyzed against PBS. The concentrations of the purified proteins were determined by the Bradford assay using BSA as a standard.

An S. exigua inhibitor of apoptosis protein gene (Seiap) was amplified from the cDNA of HvAV-3h-infected S. exigua larvae according to a previously described method (54 (link)). Four S. exigua caspase genes (SeCasp-1, SeCasp-6, SeCasp-7, and SeCasp-8) reported in our previous study were amplified from cDNA using specific primers (Table 1) (67 (link)). The five genes were then subcloned into the multiple cloning sites of the pET-28a(+) vector (Novogene, Beijing, China), followed by induction of expression in Escherichia coli strain BL21(DE3). The expressed 6×His tag-fused SeCasps and SeIAP were affinity purified with cOmplete His tag purification resin (Roche, Basel, Switzerland). Protein samples collected during the purification procedures were separated, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein (with purity of >95%) was sent to the Wuhan Institute of Virology, Chinese Academy of Sciences, to prepare rabbit polyclonal antiserum.