Strep tactin superflow

Expression and purification were performed in variations of established protocols as previously reported^{46 ,57 (link)}. In short, proteins were produced in E.coli BL21Star (DE3) or BL21 (DE3) cod+ and purified depending on the affinity tag. For in in vivo biotinylation on the Avi-tag the plasmid pBirA (Avidity Nanomedicines, La Jolla, CA) was cotransformed and the cell culture procedure was adapted according to Avidity’s in vivo biotinylation protocol.
His₆-SUMO tagged Hsp90 was isolated with affinity chromatography using a Ni-IMAC column (HisTrap HP), followed by SUMO cleavage with SenP protease and a second Ni-IMAC to separate the His-tag free protein from uncleaved proteins and the His₆-SUMO-tag. The protein was then applied to an anion exchange column (HiTrap Q, Cytiva) and finally to a size exclusion column (Superdex 200, Cytiva).
Strep-tagII and simple His₆-tag preparations were performed with a two-step protocol, starting with affinity chromatography using either a Strep-Tactin column (Strep-Tactin Superflow, IBA Lifesciences) or a Ni-IMAC (Cytiva/ Ni-NTA Agarose, Qiagen) followed by Size exclusion chromatography (Superdex 200, Cytiva).

Recombinant, soluble EphA2 Fc/Strep-fusion protein was purified under native conditions by Strep-Tactin chromatography from 293T cell culture supernatant. 293T cells were transfected by Polyethylenimine "Max" (PEI) (Polysciences) [67 ] transfection with pEphA2-Fc. The protein-containing cell culture supernatant was filtered through 0.22μm PES membranes (Millipore) and passed over 0.5ml of a Strep-Tactin Superflow (IBA Lifesciences) matrix in a gravity flow Omniprep column (BioRad). Bound protein was washed with approximately 50ml phosphate buffered saline pH 7.4 (PBS) and eluted in 1ml fractions with 3mM desthiobiotin (Sigma-Aldrich) in PBS. Protein-containing fractions were pooled and buffer exchange to PBS via VivaSpin columns (Sartorius) was performed. Protein concentration was determined by absorbance at 280nm. Aliquots were frozen and stored at −80°C. Recombinant, human, soluble EphB3-Fc (5667-B3-050) and soluble ephrin ligands, as either human (rh) or mouse (rm) Fc-fusion proteins (rm-ephrinA1, rm-ephrinA2, rh-ephrinA3, rh-ephrinA4, rh-ephrinA5, rm-ephrinB1, rm-ephrinB2 and rh-ephrinB3 Fc) (SMPK3) were purchased from R&D Systems.

Full length human IRF4 and mouse IRF8 were cloned into a pUC19 based plasmid with T7 promoter and T7 terminator containing N-terminal strep-tag followed by cleavage site for thrombin protease as described (39). The construct was transformed into Escherichia coli BL21(DE3) and grown in LURIA BROTH (Sigma). Protein expression was induced by adding 0.4 mM isopropyl-B-thiogalactoside (IPTG) for 3 h at 30 °C. The proteins were purified using Strep-Tactin Superflow (IBA Life Sciences) following the manufacturer’s instructions. The strep-tag was cleaved off by thrombin protease digestion for 8 h at room temperature.