Strep tactin superflow plus

For Strep, Myc and FLAG pulldown experiments proteins were immobilized using either Strep-Tactin Superflow Plus (QIAGEN), Anti-c-Myc Agarose (Thermo Fisher) or Anti-FLAG M2 affinity resin (Sigma). Proteins were added and incubated with the resin for 15 minutes at room temperature. Beads were washed three times with 15-20 bed volumes each. Bound proteins were eluted using either 2.5 mM desthiobiotin (Sigma), 500 μg/ml Myc peptide (GenScript) or 100 μg/ml 3X FLAG peptide (Sigma) and analyzed by SDS-PAGE.

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/streptomycin (P/S) for cell culture, blocking solution for Western blot, MANT-ADP for ATPase binding assay, Opti-MEM medium, siRNA (HSP90β, scramble), lipofectamin 2000, lipofectamine RNAi Max for transfection, qRT-PCR kit were obtained from Life Technology. RIPA lysis buffer, ECL were purchased from Thermo; protease inhibitors and phosphor-stop were acquired from Roche. Strep-tactin superflow plus were purchased from Qiagen. Bradford protein assay kit was purchased from Bio-rad. High Throughput Colorimetric ATPase Assays kit were from Innova Biosciences. Caspase-3 activity assay kit was purchased from Cell Signaling. Apoptosis detection kit was purchased from BD. For antibodies, Raf-1, IKK-1/2, HSP90β, p-HSP90β, HSP70, CDC37, PPIA, p-MEK, MEK, p-ERK, ERK, GAPDH, PARP and Ub antibodies were purchased from Santa Cruz; Anti-flag antibodies was from Sigma; CDK4, p-Rb, Caspase-9, eEF2 and Strep-HRP antibodies were from Cell Signaling. All chemicals not listed above were purchased from Sigma-Aldrich.

The purification procedures for full-length human RAD51AP1, the RAD51AP1 fragments and mutants, and for human RAD54, RAD51 and NAP1 have been described elsewhere (5 (link), 6 (link), 11 (link), 24 (link), 41 (link), 42 ). Purified human histone octamer was purchased from The Histone Source at Colorado State University (https://www.histonesource.com/) and purified as described (43 (link)).
For purification of the RAD51AP1-N88 protein, E. coli Rosetta cells harboring the plasmid were grown and induced for protein expression as described earlier (42 ). RAD51AP1-N88 was purified by tandem purification using Ni-NTA resin (Thermo Scientific) first, as described (42 ). Eluted protein was incubated with pre-equilibrated Strep-Tactin Superflow Plus (Qiagen) and gentle rotation at 4 °C for 1 h in binding buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM DTT. The resin was washed 4 × in three bed volumes of binding buffer, bound protein was eluted with binding buffer containing 2.5 mM d-Desthiobiotin (Millipore) and dialyzed into dialysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.05% Triton X-100, 2 mM DTT and 20% glycerol) at 4 °C overnight before snap-freezing, and stored at –80 °C.

As previously described, plasmids encoding mammalian codon-optimized RSV F prefusion (DS-Cav1) and postfusion (FΔFP) proteins were used to transfect Expi 293F cells (Life Technologies), and proteins were purified from culture supernatants [13 (link), 21 (link)]. Briefly, cell culture supernatants were harvested day 3 (FΔFP) or 7 (DS-Cav1) post-plasmid transfection, and RSV F proteins were purified using Ni-Sepharose chromatography (GE Healthcare). FΔFP was further purified by Strep-Tactin chromatography (Strep-Tactin Superflow Plus, Qiagen). Tags were removed from DS-Cav1 and FΔFP by overnight digestion with thrombin. To remove IMAC contaminants and uncleaved F protein, DS-Cav1 was subjected to a second Ni-Sepharose chromatography step. Both DS-Cav1 and FΔFP were polished by gel filtration chromatography (Superdex 200, GE Healthcare) and were stored in a buffer of 50 mM HEPES pH 7.5, 300 mM NaCl.

Both domains were synthesized in E. coli Rosetta 2(DE3)(pLysSRARE2). Harvested cells were resuspended in 40 mM triethanolamine (pH 8) buffer containing 400 mM NaCl and frozen at −20 °C for at least 24 h. Afterwards, cells suspensions were incubated for 30 min at 37 °C. Simultaneously, 30 mM triethanolamine (pH 8), 30 mM NaCl, 6 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, and 10 U/ml benzonase were added together with approximately 4000 U/mL lysozyme. The cells were disrupted by brief sonication and the crude extracts were obtained by centrifugation. The Strep-tag II containing target proteins were purified from the crude extract by affinity chromatography using Strep-Tactin columns (Strep-Tactin superflow Plus, 1-ml bed volume; Qiagen, Hilden, Germany) according to supplier’s instruction and concentrated in ultrafiltration devices (Sartorius Stedim Biotech, Göttingen, Germany) with a molecular mass cut-off at 10 kDa. For storage at 4 °C, 10% (v/v) glycerol was added. The protein concentration was determined after Bradford44 (link) with bovine serum albumin (BSA) as the standard and the purity was subsequently analysed by SDS-polyacrylamide gel electrophoresis45 (link).

A SalI-SacI fragment containing coding sequence for EcoKMcrB-N [18 (link)] was synthesized (GeneWiz) and cloned into the pET52 Expression Vector (Millipore) and transformed into the T7 Express cell line (NEB). The expressed recombinant protein has an N-teminal Strep tag and a C-terminal His tag from the pET52 vector to facilitate purification. Cultures were propagated at 37°C until OD₆₀₀ is 0.4–0.6 and induced with IPTG at a final concentration of 0.05 mM. Induction was performed at 30°C on shaker for 4 hours and the cells were harvested by centrifugation. Lysates were prepared by Lysozyme treatment on ice and freeze-thaw. Lysates were clarified by centrifugation for 30 minutes followed by purification with Strep-Tactin Superflow Plus (Qiagen).
The tagged McrB-N was biotin labeled with the EZ-Link Sulfo-NHS-biotin kit (Pierce, Rockford, IL) following the manufacturer’s instructions. The extent of biotinylation was evaluated using the HABA assay (Pierce). Each mole of the tagged McrB-N was found to contain 6 mole of biotin.