Bacterial strains and growth conditions
E. coli DH5α and E. coli BL21*DE3 were purchased from Invitrogen and Novagen, respectively. E. coli DH5α was used for cloning and plasmid preparation. E. coli BL21*DE3 dsbA::aph was constructed by P1 transduction from E. coli TOP10 dsbA::aph . E. coli BL21*DE3 dsbA::aph was routinely cultured in Lysogeny Broth (LB), 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl, and used for recombinant protein production. For batch fermentation E. coli BL21*DE3 dsbA::aph was cultured in complex media composed of 45 g/L yeast extract, 10 g/L glycerol, 16 g/L K2PO4 and 4 g/L KH2PO4 in scaled-up conditions. If required, culture media were supplemented with the appropriate antibiotics and inducers as required: ampicillin (100 μg/mL), kanamycin (25 μg/mL) and IPTG (0.5 mM).
SDS-PAGE and Immunoblotting
Sample Buffer Laemmli 2X concentrate (Sigma) and 2 μL of DTT 1 M (Invitrogen) were mixed with protein samples prior to incubation at 100℃ for 5 minutes. When a non-reducing and non-denaturing analysis was required, samples were treated as specified before but omitting DTT and heating. Samples were loaded onto a pre-casted 4-12% SDS-PAGE protein gel (Invitrogen) with PageRuler Plus Prestained Protein Ladder (Thermo Scientific). Proteins were detected using Brilliant Blue G – Colloidal concentrate, Electrophoresis Reagent for protein detection (Sigma) as per the manufacturer’s instructions. Specific proteins were detected by immunoblotting. In brief, proteins were separated on an SDS-PAGE gel and transferred to nitrocellulose using the iBlot Gel Transfer System (Life Technologies). The membrane was incubated with a Blotting Solution (5% w/v skimmed milk powder and PBS supplemented with 0.1% v/v Tween 20). The membrane was washed three times with PBST (PBS supplemented with 0.1% v/v Tween 20) and incubated with a dilution of the appropriate primary antibody in Blotting Solution. A 1:5,000 dilution of a mouse monoclonal antibody raised against poly-histidine tag was used. The membrane was washed three times with PBST prior to incubation with a dilution of the appropriate secondary antibody in Blotting Solution. A 1:30.000 dilution of anti-mouse alkaline phosphatase-conjugated antibodies (Sigma) was used as secondary antibodies. The membrane was washed three times with PBST prior to protein detection with BCIP/NBT-Purple Liquid Substrate System for Membranes (Thermo Fisher) or the Opti-4CN Substrate Kit (BIORAD) according to manufacturer’s instructions.
Culture supernatant protein precipitation
Culture volumes were adjusted according to the OD590 of the cultures. Cells were removed by centrifugation at 3724 x g for 20 min at 4°C. Culture supernatants were filtered through 0.22 µm filters (Millipore) and a 1:10 dilution v/v of ice-cold 100% trichloroacetic acid was added to each sample. Samples were incubated on ice for 45 min and precipitated proteins were spun down at 15,366 x g for 45 min at 4°C. Supernatants were discarded and pellets were resuspended in 1 mL of ice-cold 100% acetone. Samples were centrifuged at 18,407 x g for 1 hourat 4°C to remove acetone. The resulting pellets were resuspended in 100 μL of Sample Buffer Laemmli 2X concentrate (Sigma) supplemented with saturated Tris-solution and 1 M DTT (Invitrogen) prior to analysis by SDS-PAGE and immunoblotting.
Small scale purification of IrmA
Cultures were grown at temperatures from 30 to 37°C for small scale IrmA-S production. An aliquot of 150 mL of culture supernatant was concentrated to 3-4 mL using VivaSpin Columns (Sartorius) with 10 kDa cut-off. The concentrated culture supernatant was dialysed overnight at 4°C against Binding Buffer (50 mM Na2HPO4 pH 8, 300 mM NaCl, and 0.01% Tween 20). Samples were incubated overnight with 150 μL of Dynabeads (Life Technologies) at 4°C. In the morning, Dynabeads were washed 4 times with 1 mL of Binding Buffer and then six times with 100 µL of Elution Buffer (50 mM Na2HPO4 pH 8, 300 mM NaCl, 0.01% Tween20, and 300 mM Imidazole).
A 50 mL culture of the selected strain was inoculated from glycerol stock. The medium used was composed of 45 g/L yeast extract, 10 g/L glycerol, 16 g/L K2PO4 and 4 g/L KH2PO4 supplemented with 100 μg/mL ampicillin. A 100 mL of broth was inoculated to an OD590 of 0.2 with an overnight aerated culture and grown in a shaking incubator at 25°C at 180 rpm. A 7 litres autoclavable ADI Bioreactor (Applikon) with a working volume of 4 litres containing the medium specified above was inoculated to an OD590 of 0.05. Foam formation was prevented with PPG at a concentration of 0.75 mL/L, temperature was set at 25°C, pH was maintained at 7.2 with 4 M NaOH and 2 M H3PO4, dissolved oxygen concentration, and stirrer speed were controlled with the Bioxpert software (Applikon). The fermenter culture was induced at OD600 12.5 for 3 h with 0.5 mM IPTG. Simultaneously, 30 g/L glycerol, 0.25 g/L MgSO4 and ampicillin 50 mg/L were added. Bacteria were pelleted by centrifugation at 12,000 x g for 1 h at 4°C to remove cell debris and particulates. Subsequently, the supernatant fraction was filter sterilized through a 0.22 μm filtering unit (Corning) and kept at 4°C prior to further analysis.
Scaled-up purification of IrmA
Two litres of fermentation supernatant were concentrated 20-fold at 4°C by tangential flow filtration using a Sartocon Slice 200 (200 cm2 filter area, 10-kDa cut-off, hydrosart membrane, Sartorius). The retentate containing IrmA-S was then diafiltrated against 20 volumes of PBS. During the diafiltration step, the retentate volume was kept constant, the input pressure was maintained at 1.8–2.0 bar and transmembrane pressure at 1.1–1.2 bar. The retentate was collected for further purification of IrmA-S.
The tangential flow filtration retentate was purified by affinity chromatography using an ÄKTA Purifier 10 (GE, Healthcare) and a 1 mL HisTrap HP Column (GE Healthcare). Protein absorbance at 214, 260 and 280 nm was monitored. The mobile phase (Binding buffer HP) was composed of 50 mM Na2HPO4 pH 8, and 300 mM NaCl and was used to equilibrate the column with a 1 mL/min flow rate in 10 column volumes. The culture supernatant was loaded onto the column at 1 mL/min flow rate. The column was washed with 5 column volumes of Binding buffer HP. The IrmA-S was eluted with Elution buffer HP (50 mM Na2HPO4 pH 8, 300 mM NaCl, and 400 mM imidazole) at 1 mL/min flow rate in 10 column volumes. One mL fractions were collected at every purification step for further analysis. If required, the fractions containing IrmA-S were pooled and dialysed against 2 L of Binding Buffer HP overnight at 4°C. Proteins were quantified by the Micro BCA Assay using the Micro BCA Protein Assay Kit (Thermo Scientific).Affinity chromatography fractions containing the antigen IrmA-S were combined. Salt was removed by buffer exchange chromatography. A Sephadex G-25 column HiPrep 26/10 Desalting (GE Healthcare) was equilibrated with 50 mM Tris-HCl pH 8.0 at a 4 mL/min flow rate. The sample was loaded on the column and eluted isocratically at a 4 mL/min flow rate for 1.5 column volumes. Fractions of the void volume were pooled and subjected to further purification by ion exchange chromatography. An HiTrap Q Sepharose FF column (GE Healthcare) was used. Protein absorbance at 215, 260 and 280 nm was detected. The column was equilibrated with 50 mM Tris-HCl, pH 8.0 (Binding buffer) at a 1 mL/min flow rate. Fractions were loaded on the column with a 1 mL/min flow rate. The column was washed with 5 column volumes of Binding buffer. IrmA-S was eluted with a linear gradient from 0 to 100% of NaCl 500 mM in 20 column volumes of Binding buffer. One mL fractions were collected and analysed by SDS-PAGE to determine the fractions containing IrmA-S.
Differential scanning calorimetry
A MicroCal VP-Capillary DSC instrument (GE Healthcare) with integrated autosampler was used for Differential scanning calorimetry (DSC). In brief, DSC scans were in the temperature range of 10-130°C with a thermal ramping of 150°C per h and a 5 s filter period. Samples were exchanged in PBS pH 7.4. For each sample 500 μL at a concentration of 12 μM (estimated by Nanodrop) were analysed; the samples were transferred to a 96-well plate and left in the instrument autosampler at 5°C until analysis. A 30 μM lysozyme solution was analysed at the beginning and at the end of the set of experiments to confirm a high degree of reproducibility of the data collected (Tm = 71.2 ± 0.1). Data were analysed using the Origin 7 software (OriginLab), subtracting the data recorded for a sample containing only buffer to the reference data.
Reverse-phase high pressure liquid chromatography
IrmA-C and IrmA-S were analysed by reverse-phase high pressure liquid chromatography (RP-HPLC) with a UPLC system Acquity-UPLC (Waters). Samples were analysed on a BEH C4 Column 300Å (2,1 x 150 mm, 1,7 μm particles size, Acuity UPLC Protein). The mobile phase was composed by buffer A (0.1% TFA) and buffer B (0.1% TFA, 90% acetonitrile). Samples were eluted at a flow rate of 4 mL/min in three steps: with 97.7% buffer A and 2.3% buffer B for 2.5 min; with 0% buffer A and 100% buffer B for 10 min; and finally with 97.7% buffer A and 2.3% buffer B for 2.5 min, at 0.4 mL/min flow rate. Protein absorbance at 215, 260 and 280 nm was measured.