Sigmafast protease inhibitor

At 70–80% confluence in a T-75 flask, the CM was harvested from all the cell lines and centrifuged at 14,000× g for 15 min at 4 °C, to remove insoluble debris. A 4-mL aliquot was concentrated to 1 mL volume into GyroVap and desalted using PD-10 Desalting Columns (GE Healthcare). The CM were poured into 15-mL tubes, frozen and lyophilized to dryness. The samples were resuspended with 50 µL of RIPA buffer containing 1× Sigma FAST Protease Inhibitors. Samples were pre-cleared with 50% G-Sepharose beads in RIPA buffer + protease inhibitors for 2 h at 4 °C on an orbital shaker. Beads were removed by centrifugation at 10,000 rpm for 1 min. The c10orf118 antibody (2.5 µg) was added to the sample and incubated at 4 °C overnight on an orbital shaker. To collect the immunocomplex, G-Sepharose beads were added to the sample and incubated at 4 °C overnight. All immunoprecipitates were washed five times with RIPA buffer. Beads were boiled in 3× sample buffer for 5 min at 95 °C and centrifuged. Eluates were analysed with SDS-PAGE and western blot.

Female Golden Syrian hamsters aged 12–16 weeks, weighing approximately 150 g were purchased from Janvier Labs and housed in IVCs. Hamsters were randomly divided into 4 groups: Experimental group given capsules containing recombinant CD0873 in excipient (n = 4), experimental group given capsules containing recombinant CDTcdB-RBD in excipient (n = 4), non-immunised i.e., naïve negative control group (n = 2) and a group given capsules containing excipient only (n = 2). The purpose of this latter group was to check if the capsule or excipient contributed towards any immunogenicity detected in experimental groups. Oral dosing with capsules was performed on days 1, 15 and 30. Hamsters were euthanised 14 days post final immunisation (2.16). Blood was collected by cardiac puncture, left to clot overnight at 4 °C and serum harvested after centrifugation. A section of the ileum was fixed in 10% (v/v) neutral buffered formalin (NBF) (Sigma) for histological analysis (2.15). The remainder of the small intestine was placed in 5 mL PBS containing SIGMAFAST™ protease inhibitors (Sigma ), flushed through twice with 1 mL of this suspension then the supernatant collected after centrifugation. Serum and intestinal fluid were filter-sterilised and stored at −80 °C.

Whole cell lysate was prepared using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH8.0, SIGMAFAST™ Protease Inhibitors (Sigma-Aldrich, Cat# S8830-20TAB) and PhosSTOP EASYpack phosphatase inhibitor cocktail (Roche, Cat# 4906837001). Protein concentration was determined by Bradford assay (Bio-Rad, Cat# 5000006). 50–30 μg total proteins were separated by 7.5% or 10% tris–glycine polyacrylamide gel, with overnight incubation with primary antibodies (Table S1) and followed by a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP signals were detected using Western Lightning® Ultra Chemiluminescence Substrate (PerkinElmer INC., Cat# NEL113001EA) and images captured by a UVP ChemStudio Plus BioImaging system. The densitometry of blot bands was quantified using Image Lab 6.0 software.

Expression plasmid pPOPINF containing ORF for cTXNPx from L. amazonensis (AY842247.1) tagged with N-terminal 6xHIS-tag was transformed into BL21 (DE3) competent cells (Agilent Technologies). One litre LB-ampicillin cultures were induced to express recombinant protein by addition of 1M isopropyl β-D-1-thiogalactopyranoside (IPTG). After 20 h of incubation at 30°C, cells were pelleted and resuspended in 50 mL of lysis buffer (20 mM Tris-HCI pH 7.5, 500 mM NaCI, 30 mM imidazole and 2 mM β-mercaptoethanol) with one tablet of Sigma-Fast protease inhibitors (Sigma-Aldrich). Cell pellets were further lysed by sonication and proteins were purified using His-tag affinity chromatography (HisTrap HP-1 mL column, GE healthcare). The cTXNPx peak fractions were pooled and concentrated to approximately 1 mg/mL as determined spectrophotometrically using a NanoDrop at 280 nm.

Restriction enzyme digests, DNA ligations, and other recombinant DNA procedures were performed using standard protocols. Mutagenesis was performed using the QuikChange site-directed mutagenesis method (Stratagene) with the KOD polymerase (Novagen). DNA constructs used for transfection were purified from E. coli DH5α using Qiagen Plasmid kits according to the manufacturer’s protocol. All DNA constructs were sequence verified. Lysis buffer used for purifying proteins from insect cells was composed of 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 20 mM imidazole, 5% (v/v) glycerol, 0.075% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 1 mM PMSF. Lysis buffer was supplemented with SigmaFast protease inhibitors (EDTA-free) at 1 tablet/100 ml (Sigma). Wash buffer A was the same as lysis buffer without PMSF and protease inhibitors. High salt wash buffer B was the same as buffer A with 500 mM NaCl. All protein concentrations were calculated using the Beer-Lambert law by measuring the absorbance at 280 nm. Theoretical extinction coefficients were calculated using the ProtParam tool at the ExPASy proteomics server (http://www.expasy.org).
See Supplementary Information for a detailed description of the methods and materials used in this study.

The indirect hydroxylation assay was performed as reported previously^{4 (link),31} . Briefly, 4 μg of biotinylated HIF2α-CODD peptide was immobilized on streptavidin-agarose beads. Following immobilization, the beads were washed 2× with EBC buffer (50 mM Tris–HCl, 120 mM NaCl, 0.5% (v/v) NP-40) and then 2× with 50 mM Tris–HCl pH 7.5. The immobilized peptides were then incubated with 7.5 μg HIS₆-PHD2 (residues 181–426) in 500 μl of 50 mM Tris–HCl pH 7.5, 300 μM α-ketoglutarate, 2 mM ascorbic acid, 1 mM dithiothreitol, and 150 μM FeCl₂ at room temperature for 1.5 h with gentle agitation. The beads were washed 5× with EBC buffer. Immobilized peptides were then incubated with HA-pVHL₃₀, produced via in vitro transcription and translation in rabbit reticulocyte lysate (Promega), at 4 °C for 1.5 h in EBC buffer with 1× EDTA-free SigmaFast protease inhibitor (Sigma-Aldrich). The beads were again washed 5× with EBC buffer. Immobilized material was boiled in 1× SDS-PAGE sample buffer to elute protein and HA-pVHL₃₀ protein amounts were examined via Western blot as a surrogate for hydroxylation.