Factor xa

Purification of the N-terminal domain of aralar in the calcium-free state was identical to the purification procedure of the calcium-bound state until the tag removal step. The eluted sample was incubated for 15 min with 50 mM EDTA, pH 8.0 and 50 mM EGTA, pH 7.5. EDTA and EGTA were removed from the sample by diluting in lysis buffer followed by ultrafiltration with a 30-kDa MWCO centrifugal concentrator. The sample was concentrated to 1 ml and 50 μg Factor Xa (New England Biolabs) was added for tag removal overnight. 10 mM EDTA and 10 mM EGTA were added to the sample and incubated for 15 min on ice. The sample was injected onto a Superdex 200 HiLoad 16/60 120 pg column at 1 ml min⁻¹ with SEC buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, pH 8.0 and 10 mM EGTA, pH 7.5). Peak fractions were pooled and concentrated to 7.5 mg ml⁻¹ in a 30-kDa MWCO centrifugal concentrator. The protein concentration was determined using the NanoDrop ND-1000 (Thermo Scientific) with parameters determined using ExPasy ProtParam64 . Purified protein was flash frozen and stored in liquid nitrogen.

The pMal-p2x vector includes a Factor Xa protease sequence allowing the scFv to be cleaved from MBP after purification. The fusion protein was incubated at 20°C for 16 hours with Factor Xa (New England Biolabs, UK) and the products separated by HiTrapQ (GE Healthcare, UK) anion exchange chromatography eluted with a gradient of NaCl (0 to 0.5 M). A linear gradient resulted in poor separation (the peaks were overlapping) and so, as each protein peak started to elute, the NaCl concentration was held until the signal returned to baseline. This method allowed complete separation of the free scFv from both MBP and the uncleaved fusion protein.

MG132(calbiochem), chloroquine(CQ; Sigma-Aldrich), polybrene(Sigma-Aldrich), lipofectamine 2000(Invitrogen), puromycin(InvivoGen), EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail(Roche Diagnostics), mouse monoclonal M2 FLAG affinity gel beads(Sigma-Aldrich), ammonium bicarbonate(NH₄HCO₃; Sigma-Aldrich), iodoacetamide(IAM; Sigma-Aldrich), formic acid(Sigma-Aldrich), Trifluoroacetic acid(TFA; Pierce), tris(2-carboxyethyl)phosphine(TCEP; Pierce), bovine trypsin(Roche Applied Science), acetonitrile(ACN;Thermo Fisher Scientific), POROS 20 R2 beads(Applied Biosystems), C18 ZipTips(Merck Millipore), isopropyl-β-D-thiogalactopyranoside(IPTG; Sigma-Aldrich), Dynabeads protein G(Invitrogen), protein G Sepharose(GE Healthcare Life Sciences), NuPAGE Bis-Tris and Tris-Acetate gels running system(Invitrogen), QuikChange Lightning Site-Directed Mutagenesis Kit(Agilent Technologies), Hybond-P PVDF membrane(GE Healthcare Life Sciences), BCA Protein Assay Reagent Kit(Pierce), Radioactive [γ-³²P]ATP(PerkinElmer), Calf intestinal alkaline phosphatase(New England Biolabs), Factor Xa(New England Biolabs), K48- and K63-linked poly ubiquitin chains(Boston Biochemical), DAPI-containing fluorescence mounting medium(Invitrogen) were purchased from indicated suppliers.

GST-fused and MBP-fused proteins were purified from E. Coli by affinity chromatography with Glutathione Sepharose 4B and amylose resin, respectively, as described in the instruction manuals (GE Healthcare and New England Biolabs, respectively). Purified GST-fused and MBP-fused proteins were dialyzed using Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) and were concentrated by Amicon Ultra Centrifugal filter devices (Millipore, Billerica, MA) and were separated by electrophoresis on a 10% SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining (Simply Blue SafeStain, Thermo Fisher Scientific).
For direct interaction studies, MBP-fused proteins were incubated with amylose resin at 4°C for 2 hours and washed with PBS. Purified GST-fused proteins were added to amylose-resin-bound MBP-fused proteins, and the mixture was incubated overnight at 4°C. After washing, the bound proteins were eluted with NuPAGE LDS sample buffer at 99°C for 10 minutes.
Twenty five microgram of purified MBP-LZTFL1 fusion protein was cleaved in 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂ (pH 8.0) containing 1 μg of Factor Xa (New England Biolabs) for 6 hours at room temperature and used for direct interaction studies with purified TfR1-Myc-Flag protein (TP326147, OriGene) conjugated anti-TfR1 antibody beads or GST-fused β1subunit of AP-1 conjugated Glutathione Sepharose 4B.

30 uL of tagged Fluc-WT proteins were incubated with Factor Xa (NEW ENGLAND BioLabs, final concentration 67 ug/mL) for 2 hours or overnight on ice.