Histrap ff crude

Stable cells expressing HBRV Su were expanded to 12 × 15 cm cell cultural dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The medium in each plate was replaced with 25 ml Pro293™ CD serum-free medium (Lonza) when cells reached 95% confluence. The medium was collected after 5-6 days of incubation and centrifuged at 3,000 g for 20 min. The clarified medium was adjusted to pH 8.0 and filtered through a 0.22 μm filter before purification.
Purification of HBRV Su was performed on 1 ml Histrap FF crude column and buffers as suggested by the supplier (GE Healthcare) using an ÄKTA explorer 100 (Amersham Pharmacia Biotech). The conditioned medium was loaded to the equilibrated column at the rate of 1 ml/min, and the column was then washed with 20 ml binding buffer and eluted into 10 × 0.5 ml fractions using elution buffer. The peak elution fraction was combined and changed to proteins storage buffer by ultrafiltration (Millipore, 30 kDa cutoff limit concentrator, 4000 g for 20 min). The final preparation was aliquoted for storage at −80°C for ELISA. The 10 eluted fractions were assessed by western blot analysis using anti-MMTV Env antibody or anti-FLAG antibody and 10% SDS-PAGE gels stained with Coomassie R-250 blue stain (Bio-Rad). The protein concentration was determined by BCA assay (Pierce) using bovine serum albumin (BSA) as a standard.

Full-length mouse TRF1 was cloned using the Gateway^™ technology (Thermo Fisher Scientific) into the expression vector pDEST565, which adds two tags (6xHis and GST) at the N terminus of the encoded protein. Protein was expressed in Escherichia coli strain BL21(DE3) and purified by affinity chromatography using a Ni²⁺ column (HisTrap FF crude, 17-5286-01, GE Healthcare) in an AKTA Prime system (GE Healthcare), followed by dialysis against 20 mM phosphate buffer pH 7.5, 200 mM NaCl, 2 mM TCEP.
One μg of purified GST-TRF1 was incubated with 0.2 μg of human GST-AKT1 (1379-0000-2, ProQinase) with or without AKT inhibitor (MK-2206, 25 μM) in kinase buffer (50 mM Hepes pH 7.9, 100 μM ATP, 10 mM MgCl₂, 5 mM DTT) containing 5 μCi [γ-³²-P]ATP in a total volume of 25 μl. The peptide RBER-GSK3(14-27) (0349-0000-5, ProQinase) (100 ng) was used as positive control. The reactions were performed at 30 °C for 1 and stopped by addition of Lamely buffer (6×). Samples were resolved in 4–12% SDS-PAGE gels and subjected to autoradiography.

pDEST17 vector expressing His‐SlCaM6 and pMAL‐p5x vector expressing MBP‐SFI5 (28–241 aa) were transformed into Rosetta™(DE3) competent cells, respectively. Positive colonies were cultivated in LB medium at 37°C with 200 rpm shaking until the culture reached an OD₆₀₀ of 0.6, followed by addition of 0.5 mM isopropyl β‐d‐thiogalactopyranoside (IPTG) for protein induction. Upon induction, bacterial cultures were transferred to 28°C with 200 rpm shaking for 2 h before harvesting. His‐ and MBP‐fusion proteins were purified by HisTrap (HisTrap FF Crude; GE Healthcare, Freiburg, Germany) and MBPTrap (MBPTrap HP; GE Healthcare) columns, respectively, following the user manuals. For pull‐down assay, the same amount (5 mmol) of His‐SlCaM6 and MBP‐SFI5 were mixed together with 5 mM CaCl₂ or 5 mM EGTA in 500 μl PBS buffer and incubated at 4°C for 1 h. Both protein mixtures were then incubated with 10 μl Ni‐NTA resin at 4°C for 30 min, followed by the removal of the supernatant and extensive wash (eight times) with wash buffer (250 mM This‐HCl, 500 mM NaCl, 25% (v/v) glycerol, 5 mM β‐mercaptoethanol, and 10 mM imidazole. pH 8.0). The binding proteins were eluted with 1 M imidazole and mixed with SDS sample buffer for SDS‐PAGE. Purified His‐SlCaM6 and MBP‐SFI5 proteins also were loaded for SDS‐PAGE as controls.

Cloning and protein purification were performed as described previously (Käppel et al. 2018 (link)). Here is just a short summary of the strategy.
SEPALLATA3 (SEP3) cDNA (GenBank accession NM_102272, positions 1–270 of the CDS) was cloned into the bacterial expression vector pET-15b (Merck Millipore), thereby creating an N-terminal fused His₆-tagged protein. The fragment contains DNA encoding the MADS- and the I-domain and was therefore termed SEP3_MI. SEP3 mutants encoding proteins with single amino acid exchanges (SEP3_MI R3A and SEP3_MI R3K) were created by site-directed mutagenesis from the previously created vector carrying SEP3_MI.
Protein purification was done with the help of an Äkta purifier 10. First, the His₆-tagged proteins were purified on a Ni sepharose column (His-Trap FF crude, GE Healthcare). Bacterial DNA was removed by employing a heparin sepharose column (HiTrap Heparin HP, GE Healthcare). Then the His₆-tag was removed by thrombin cleavage. Proteins were finally purified using size exclusion chromatography (Superdex75 10/300 GL column, GE Healthcare).

E. coli BL21(DE3) were transformed with the E2 expression plasmid and grown in TY medium (1% peptone, 0.7% yeast extract, 0.25% NaCl) shaking at 37 °C for 16 h. Precultures were diluted in TY medium and further incubated shaking until induction with 1 mM IPTG, at OD₆₀₀ 1.0, executed at 37 °C, 220 rpm, for 4 h. Cell pellets were harvested by centrifugation, and stored at −20 °C until purification.
Frozen cell pellets were resuspended in Tris-buffer (50 mM Tris/HCl, 50 mM NaCl, pH 7.5), disrupted with a French press and the clarified supernatant applied to an IMAC column (HisTrap FF Crude, 1 mL, GE Healthcare, Uppsala, Sweden) connected to an ÄKTA purifier system. Washing was executed for five column volumes with running buffer (50 mM Tris/HCl, pH 7.4, 300 mM NaCl, 20 mM Imidazole). Elution was conducted for five CV with the same buffer containing 250 mM imidazole.
After affinity chromatography imidazole and excess NaCl from pooled peak fractions were exchanged to Tris-buffer with a gel filtration column (HiTrap Desalting, 5 mL, GE Healthcare, Uppsala, Sweden) connected to an ÄKTA purifier system. All elution fractions were pooled, the concentration determined with a BCA assay (Bicinchoninic acid assay, Pierce; BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA), aliquoted, and stored in Tris-buffer with 2 mM DTT at −80 °C.