Actev protease

GB1-CTB proteins (2 μg) purified from the plant extracts were mixed with 0.2 U AcTEV™ Protease (Invitrogen, Cat. 2575015) and 2 μL 20 x TEV buffer, by adding ddH₂O up to 40 μL. The mixtures with or without TEV protease were incubated at 10 or 25^°C overnight, followed by SDS-PAGE analysis.

The cell lines samples were prepared for IP as indicated in the “Immunoblotting” section. For purification of CERS6 (Fig. 5a), 2000 µg of protein lysates as prepared above were pulled down at 4 °C overnight with Ni-NTA His-Bind Resin (EMD-Millipore, Billerica, MA), washed 4 times with modified RIPA and then eluted with 350 mM imidazole. Further, the eluate was incubated with EZview Red Streptavidin Affinity Gel (Sigma) for 2 h, washed 4 times with modified RIPA, and digested with AcTEV Protease (Invitrogen, Carlsbad, CA) for 3 h. Immunoprecipitation of Fas was performed using 1000 µg of protein lysates incubated with 2 µg of anti-Fas antibody Rb (Proteintech, Rosemont, IL) overnight at 4 °C. Fas and associated proteins were pulled down using 50 µl of Protein G Plus Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and sample prepared using 4× NuPAGE lithium dodecyl sulfate loading buffer and 100 mM DTT, followed by heating at 70 °C for 10 min. For immunoprecipitation of CERS6 (Fig. 6b), 1000 µg of protein lysates were pulled down at 4 °C overnight with 50 µl EZview Red anti-FLAG-M2 affinity gels (Sigma-Aldrich), washed 4 times with modified RIPA and then eluted with an excess of 3× FLAG peptide (100 μg/ml). Immuno-complexes were resolved by 4–12% SDS-PAGE and immunoblotted with the indicated antibodies.

Total chromosomes (10⁷) or chromosome 19 (2 × 10⁵) were incubated with or without the AcTEV protease (Invitrogen, 10 units) for 4 h at RT with gentle rotation, according to the manufacturer’s instructions. Samples were then collected for western blot, or prepared for optical imaging or Cryo-ET.

His-tagged chicken FANCI and FANCD2 were purified exactly as described previously (Rajendra et al., 2014 (link)). Recombinant His-Strep-HA-RNF4 was produced by the CPR Protein Production Unit, and the His-Strep-HA tag was removed by AcTEV protease (Invitrogen) according to the manufacturer’s instructions. For in vitro SUMOylation assays, components (SAE1/2, UBC9, PIAS1, SUMO2, FANCI, or FANCD2) were added to a total reaction volume of 30 μl in SUMOylation buffer (50 mM Tris [pH 7.5], 5 mM MgCl₂, 0.6 mM dithiothreitol, 2 mM NaF) and incubated at 30°C for 2 hr. For STUbL assays, in vitro SUMOylation reactions scaled up 3-fold were diluted in 500 μl binding buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 0.05% NP-40, 1 mM imidazole) and added to Ni²⁺ agarose for 2 hr at 4°C. Bound proteins were washed extensively in 50 mM Tris (pH 7.5), subjected to in vitro ubiquitylation by recombinant RNF4 at 37°C for 90 min, washed again, and analyzed with immunoblotting.

Recombinant Pum and Nos for EMSAs were expressed in KRX E. coli cells (Promega) in 2xYT media with 25 µg/mL Kanamycin and 2 mM MgSO₄ at 37°C to OD₆₀₀ of 0.7–0.9, at which point protein expression was induced with 0.1% (w/v) rhamnose for 3 hr. Cell pellets were washed with 50 mM Tris-HCl, pH 8.0, 10% [w/v] sucrose and pelleted again. Pellets were suspended in 25 mL of 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 0.05% (v/v) Igepal CA-630, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin. To lyse cells, lysozyme was added to a final concentration of 0.5 mg/mL and cells were incubated at 4°C for 30 min with gentle rocking. MgCl₂ was increased to 7 mM and DNase I (Roche) was added to 10 µg/mL followed by incubation for 20 min. Lysates were cleared at 50,000xg for 30 min at 4°C. Halo-tag containing proteins were purified using HaloLink Resin (Promega) at 4°C. Beads were washed 3 times with 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl₂, 1 M NaCl, 1 mM DTT, 0.5% [v/v] Igepal CA-630) and 3 times with Elution Buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 20% [v/v] glycerol). After washing, beads were resuspended in Elution Buffer with 30 U of AcTEV protease (Invitrogen), cleavage proceeded for 24 hr at 4°C, and beads were removed by centrifugation through a micro-spin column (Bio-Rad).

The KH23 construct was readily refolded via a three-step dialysis. The sample was first dialysed into 3 M guanidine hydrochloride, 10 mM TRIS pH 8.0, 250 mM NaCl, 0.5 mM TCEP, 0.5 mM EDTA. Before being placed in 1 M guanidine hydrochloride, 10 mM TRIS pH 8.0, 150 mM NaCl, 0.5 mM TCEP, 0.5 mM EDTA. Finally the sample was dialysed into TEV digestion buffer containing 10 mM TRIS pH 8.0, 100 mM NaCl, 1 mM TCEP, 0.5 mM EDTA.
KH23 re-folding was validated by recording 2D ¹H{¹⁵N} SOFAST-HMQC NMR spectrum.
Refolded KH23 was cleaved with AcTEV protease (Invitrogen, cat. no. 12575) following the manufacturer protocol to de-protect the N-terminal cysteine incorporated in KH2. Time points of the TEV digestion were analysed using the same HPLC conditions as the first step ligation reaction analysis. Cleaved products were analysed via microTOFQ electrospray mass spectrometer (Bruker Daltonics) Figs 2c and 3b.
Once digestion was complete the sample was purified on a pre-equilibrated C18 semipreparative reverse phase column using a 30–60% ACN gradient over 30 min. Again the purified cleaved ligated product was freeze dried and stored at −80 °C.