Strep tactin column

A membrane pellet from Strep-tagged E60 was resuspended in 20 mM Tris (pH 8), 100 mM NaCl, 1 mM EDTA to a final concentration of 50 mg/ml of total protein, followed by 1.5 g of DDM (n-dodecyl β-d-maltoside, Anatrace) per 1 g of total protein. After 1 h of incubation, the sample was ultracentrifuged for 30 min at 180,000 × g and filtered with 0.45-μm syringe filter. The sample was loaded on the Strep-Tactin column (IBA Lifesciences) preequilibrated with 20 mM Tris (pH 8), 100 mM NaCl, 1 mM EDTA, 0.05% DDM buffer. The subsequent steps were performed according to the manufacturer’s protocol (IBA Lifesciences). The total protein concentration was determined using a Pierce BCA protein assay kit. Purified protein samples were analyzed using SDS-PAGE using 4 to 20% precast gels (Bio-Rad).

Mechanical cell lysis was performed using a One Shot Cell Disruptor (Constant Systems Limited, Daventry, United Kingdom). The centrifuged biomass was thawed, resuspended with a 10-fold (w/v) mechanical lysis buffer (100 mM Tris, 150 mM NaCl, 5 mM MgCl₂, pH 8) and homogenized in the homogenizer with one shot at 1 kbar. The resulting lysate was incubated for 10 min with 25 U/mL Benzonase^® and centrifuged for 25 min at 4°C and 20,800 x g. The supernatant was filtered through a 0.2 μm filter and used as clarified E. coli cell lysate for following Strep-tag-affinity chromatography or split intein-based affinity chromatography.
To generate a Strep-purified reference sample, the clarified E. coli cell lysate was passed through a Strep-Tactin^® column (IBA Lifesciences, Göttingen, Germany) with a column volume (CV) of 132 mL, washed with equilibration buffer (100 mM Tris, 200 mM NaCl, pH 9) and eluted in 100 mM Tris, 200 mM NaCl, 2.5 mM d-Desthiobiotin, pH 9. The detailed Strep-tag affinity chromatography protocol follows the manufacturers instructions and is described in Table 1. The concentration of the proteins was determined by absorbance at 280 nm (A₂₈₀) and under consideration of the molecules specific extinction coefficient. The purified protein stock was used as a Strep-purified reference sample for following split intein-based purification.

J6 and H77 E2 ectodomains (residues 384–661) were PCR-cloned into expression vectors, as previously described (60 (link)), with the resultant constructs including an upstream tissue plasminogen activator signal sequence (to direct efficient secretion) and a downstream Strep-tag II (for detection and purification). The proteins were produced by transient transfection of HEK293T cells with the supernatants being harvested at 48 and 72 h postinfection. sE2 was purified using a Strep-Tactin column (IBA Life Sciences, Göttingen, Germany), and monomeric sE2 was subsequently isolated by size exclusion chromatography. The sE2 binding assay was performed as previously described. A single-cell suspension of CHO ± CD81 cells were preincubated in “traffic stop” buffer, described above. All subsequent steps are performed in traffic stop buffer. The cells (100 μl at 1–3 × 10⁶/ml) were then mixed with 10 μg/ml sE2 and incubated for 1 h at 37 °C. Bound sE2 was then detected using 3 μg/ml StrepMab classic followed by an anti-mouse Alexa Fluor 647 secondary (1 h of incubation each at room temperature). Finally, the cells were fixed in 1% formaldehyde. Fluorescence signals were measured by flow cytometry.

A membrane pellet from Strep-tagged E60 was resuspended in 20 mM Tris (pH 8), 100 mM NaCl, 1 mM EDTA to a final concentration of 50 mg/ml of total protein, followed by 1.5 g of DDM (n-dodecyl β-d-maltoside, Anatrace) per 1 g of total protein. After 1 h of incubation, the sample was ultracentrifuged for 30 min at 180,000 × g and filtered with 0.45-μm syringe filter. The sample was loaded on the Strep-Tactin column (IBA Lifesciences) preequilibrated with 20 mM Tris (pH 8), 100 mM NaCl, 1 mM EDTA, 0.05% DDM buffer. The subsequent steps were performed according to the manufacturer’s protocol (IBA Lifesciences). The total protein concentration was determined using a Pierce BCA protein assay kit. Purified protein samples were analyzed using SDS-PAGE using 4 to 20% precast gels (Bio-Rad).

Proteins were expressed in vivo from BL21 (DE3) strains using IPTG induction. Briefly, 1 L cultures in terrific broth (Invitrogen) supplemented with ampicillin at 100 μg/mL were grown at 37 °C to OD ~1.5 and induced with IPTG at 100 μM. At this point, the temperature was reduced to 25 °C and proteins were expressed overnight. Cultures were harvested by centrifugation at 3000 × g and cells were lysed in a single pass through a high-pressure homogenizer (Avestin) at >18,000 PSI. Debris and insoluble material were removed by centrifugation at 23,000 × g. The soluble supernatant was purified on a streptactin column (IBA Life Sciences) according to the manufacturer’s protocol. After purification, elution fractions were pooled, concentrated ~30 X using a centrifugal filter device (Amicon Ultra, 10 kDa cutoff, Millipore), formulated in 20 % sucrose, flash frozen in liquid nitrogen, and stored at − 80 °C. Typical yields were 1 mg of purified protein from 3 (wet) grams of cells.