Endosafe pts system

Purified SitC-SA and SitC-SC were tested for endotoxin contamination using the Endosafe-PTS System (Charles River, USA). Endotoxin levels for 1 µg/ml protein solutions were <0.005 EU/ml for SitC-SA and 0.006 EU/ml for SitC-SC.

The Endosafe-PTS system (Charles River, Wilmington, MA, USA) was used following the manufacturer’s instructions. The Venor qEP (Minerva Biolabs, Berlin, Germany) Kit was used for the amplification of a mycoplasma-specific 16S rRNA gene region and detection by real time polymerase chain reaction (PCR) according to the manufacturer’s instructions. In vitro adventitious agents’ detection in MSC samples were performed on MRC5 and Vero cells. If cytopathic effect was observed after 14–28 days of culture, PCR and immunofluorescence techniques were used to identify contamination agents.

DNA coding for As16 without its signal peptide was codon optimized for expression in yeast and synthesized by GenScript. The DNA coding for As14 without its signal peptide was PCR amplified with As14 specific primers from A. suum larvae cDNA reverse-transcribed from total larval RNA. As16 and As14 coding DNAs were subcloned into the yeast expression vector pPICZαA (ThermoFisher Scientific, Carlsbad). The correct sequences and reading frames of the recombinant plasmids were confirmed by double-stranded DNA sequencing using vector flanking primers, α-factor and 3’-AOX1. The recombinant As16 and As14 (rAs16 and rAs14) with a hexahistidine tag at its C-terminus were expressed in yeast stain P. pastoris X-33 under induction with 0.5% methanol for 48–72 hours and then purified by immobilized metal ion affinity chromatography (IMAC), as described previously [35 (link)]. The purity of the recombinant proteins was determined by SDS–PAGE. The protein concentration was measured using BCA (ThermoFisher Scientific, Waltham) and Endotoxin clearance was confirmed using the Charles River Endosafe-PTS system (Charles River, Houston).

Endotoxin was removed from I53-50B.4PT1 during or after purification of the protein using a detergent wash during IMAC. The protein was immobilized on a 5 mL HisTrap HP column (GE Healthcare) equilibrated with buffer (25 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS) and the column was washed with ∼10 CV of the equilibration buffer. I53-50B.4PT1 was eluted with a linear gradient of 0 to 500 mM imidazole in equilibration buffer. Fractions containing I53-50B.4PT1 were concentrated in a 10 kDa molecular weight cutoff Vivaspin filter and dialyzed twice against equilibration buffer. Purified I53-50B.4PT1 pentamer was tested for endotoxin prior to assembly using a Charles River EndoSafe® PTS system, and measured concentrations were routinely below 100 EU/mL. Before formulation in SWE or AddaVax, protein preparations were again tested to be negative for endotoxin contamination by Chromogenic Limulus Amebocyte Lysate (LAL) assay (Lonza).

The endotoxin content of the purified BMP-4 was determined by the Limulus amebocyte lysate (LAL) assay. The test was carried out using an Endosafe^®-PTS™ system (Charles River Laboratories, USA) with a cartridge ranging from 0.1–10 EU ml⁻¹ (EU = endotoxin unit) and following the manufacturer’s instructions.

DNA coding for the full-length As37 was synthesized by GenScript (Piscataway, NJ) and then subcloned into the prokaryotic expression vector pET41a using NdeI and NotI sites to exclude the GST tag. The recombinant As37 protein (rAs37) was expressed in E. coli BL21(DE3) under induction with 1 mM IPTG. Soluble rAs37 with a hexahistidine-tag at its C-terminus was purified by immobilized metal ion affinity chromatography (IMAC), as described previously [31 (link)]. Contaminating endotoxin was removed by anion exchange chromatography with a Hitrap Q HP column (GE Healthcare, Chicago, IL), while the purity of the recombinant protein was determined by SDS–PAGE and the protein concentration was measured using A280 absorption. Endotoxin clearance was confirmed using the Charles River Endosafe-PTS system (Charles River, Houston, TX).

STm phase 1 flagellin proteins (FliC) were purified as described from culture supernatants of CVD 1925 [20 (link)]. Recombinant CRM₁₉₇ produced in E. coli was obtained from Fina Biosolutions (Rockville, MD). FliC and CRM₁₉₇ were confirmed for integrity and removal of residual host cell protein by SDS-PAGE with Coomassie Brilliant Blue staining, and endotoxin levels by Limulus Amebocyte Lysate (LAL) assay using the Endosafe PTS system (Charles River Laboratories, MA).