Recombinant AcpM protein was prepared according to the previous study (19 (link)). Briefly, mycobacterial acpM was amplified from genomic DNA of Mtb H37Rv ATCC 27294 using the forward (5’-CATATGCCTGTCACTCAGGAAGAAATC-3’) and reverse primers (5’-AAGCTTCTTGGACTCGGCCTCAAGCCT-3’), and the PCR product was inserted into the pET-22b (+) vector (Novagen, Madison, WI, USA). The recombinant plasmids were transformed into E. coli BL21 cells by heat-shocking for 1 min at 42 °C. Cell disruption was used to obtain the overexpressed AcpM protein, which was then purified using NI-NTA resin. The purified recombinant protein was dialyzed and incubated with polymyxin B-agarose (Sigma Chemical Co.) to remove residual endotoxin. The purified endotoxin-free AcpM was filter sterilized and kept frozen at -80°C until use. To collect anti-AcpM antibodies, BALB/c mice were injected three times intraperitoneally with purified AcpM (25 μg per mouse) emulsified in incomplete Freund’s adjuvant. One week after the final immunization, serum was collected and stored frozen until use with proper dilution.
Polymyxin b agarose
Manufactured by Merck Group
Sourced in United States
Polymyxin B-agarose is a chromatography resin used for the purification of polymyxin-binding proteins. It consists of polymyxin B, an antibiotic, immobilized on agarose beads.
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Lab products found in correlation
Dc protein assay kit, by Bio-Rad (3 mentions)
Lal assay kit, by Lonza (2 mentions)
Lal test kit, by Lonza (2 mentions)
Endothelial growth medium, by Thermo Fisher Scientific (1 mentions)
Matrigel matrix, by Merck Group (1 mentions)
Mcp 1, by Merck Group (1 mentions)
Tetramethyl rhodamine b isothiocyanate, by Merck Group (1 mentions)
Transwell tissue culture insert, by Corning (1 mentions)
24 well plate, by Corning (1 mentions)
Calcein am, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
His select nickel affinity gel, by Merck Group (1 mentions)
Balb cj mice, by Jackson ImmunoResearch (1 mentions)
Hiload 16 600 superdex 200 pg column, by Cytiva (1 mentions)
Lal assay kit, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Lps e coli serotype o55 b5, by Merck Group (1 mentions)
Myd88, by Charles River Laboratories (1 mentions)
Female balb c mice, by Charles River Laboratories (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
19 protocols using polymyxin b agarose
1
Recombinant AcpM Protein Preparation
2
Preparation and Characterization of Schistosome Antigens
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S. japonicum SEA and SWA were prepared as previously described [21 (link),22 (link)]. The antigens were filter-sterilized and endotoxin was removed using Polymyxin B-Agarose (Sigma-Aldrich, St. Louis, MO). The endotoxin activity (<0.01 EU/μg) was determined using the LAL assay kit (BioWhittaker, Walkersville, MD). Protein concentrations were determined using the Lowry method (DC Protein Assay Kit, Bio-Rad, Hercules, CA).
3
Transwell-Based Endothelial Cell Assay
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Transwell tissue culture inserts (diameter, 6.5 mm; pore size, 5.0 µm) and 24-well plates were purchased from Corning Costar Corp (Corning, NY). RPMI 1640 medium and endothelial growth medium were obtained from Gibco (Grand Island, NY). Heat-inactivated fetal bovine serum was obtained from HyClone (Logan, Utah). Mouse anti-human claudin-1 was obtained from Cell Signaling Technology (America, Boston). The Limulus amebocyte lysate test kit, polymyxin B-agarose, anti-human ICAM-1 monoclonal antibody (MAb), anti-human E-selectin (MAb), Calcein AM, phalloidin, tetramethylrhodamine bisothiocyanate, matrigel matrix, and MCP-1 were purchased from Sigma-Aldrich (America, St Louis, MO). Recombinant T. pallidum protein Tp0965(rTp0965)was purified on nickel-nitrilotriacetic acid (Ni-NTA) chromatographic column from E. coli lysates frozen in our laboratory. The human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems, Inc. (Minneapolis, MN). Anti-mouse immunoglobulin G and horseradish peroxidase (HRP)-tagged antibody were purchased from Amersham (Piscataway, NJ). Anti-rabbit HRP-tagged antibody was purchased from Zymed (San Francisco, CA).
4
Recombinant Expression and Purification of M. tuberculosis EspC
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The gene coding M. tuberculosis EspC was amplified using the primer sequences (sense) 5′-GCGGATCCATGACGGAAAACTTGACCGTCCA-3′ and (antisense) 5′-CCCAAGCTTTCAGGTAAACAACCCGTCGATAGCCTT-3′. The espC product was digested with the restriction enzymes BamHI and HindIII, inserted into the pET28a (+) plasmid (Novagen, Madison, WI, USA), and transformed into Escherichia coli BL21 cells. Recombinant EspC was prepared by induction of bacterial cells with 0.5 mM isopropyl β-d -1-thiogalactopyranoside at 37°C for 12 h. The harvested cells were lysed as previously described. Recombinant EspC was purified using HIS-Select Nickel Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer's instructions. To remove endotoxins, the dialyzed recombinant EspC was incubated with polymyxin B-agarose (Sigma) overnight at 4°C. The preparation had a very low endotoxin content (< 0.05 EU/mg), as measured using an E-toxate (Limulus amebocyte lysate) kit (Sigma-Aldrich). The protein concentration was estimated with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA), and protein was then stored at −80°C until use.
5
Immunization Protocol with BLS Variants
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BALB/cJ mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA), bred under specific pathogen-free conditions, housed and treated according to the policies of the protocol approved by the Committee on the Ethics of Animal Experiments of the Leloir Institute following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The immunizations were performed in groups of 4–5 mice 8 to 10 weeks old, intraperitoneally, with 100 μl of phosphate saline buffer (PBS) containing 10 μg of BLSWT, BLSDRKE, BLSDR or BLSKE, previously incubated twice with polymyxin B-agarose (Sigma-Aldrich) to eliminate LPS, as previously described.22 (link) Mice sera were collected at 14, 28 and 42 d.p.i. and stored at −20 °C for future use.
6
Recombinant RBD Protein Purification
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Generating pure RBD for antibody binding assays broadly followed the method described for SARS-CoV RBD.73 (link) Pichia pastoris strain KM71H transformed with construct pPICZα-RBD encoding for ancestral SARS-CoV-2 spike, residues 333–530, was used to express and secrete the RBD. Expression was performed in BMM medium (1.34% w/v YNB, 0.00004% w/v Biotin, 1% v/v methanol, in 100 mM potassium phosphate, pH 6.0) at 30°C and 280 rpm over the course of 2-3 days with intermittent refeeding (1% v/v methanol) after 24 hours. The clarified (0.2 μm PTFE membrane) expression medium containing the secreted protein was prepared for Hydrophobic interaction chromatography by adding solid (NH4)2SO4 to 2 M. The RBD protein was captured on a HiTrap Butyl Sepharose HP column (Cytiva) and eluted using a stepwise gradient from 20 mM Tris, 2.0 M (NH4)2SO4, pH 8.0 to 20 mM Tris, 0.02 M (NH4)2SO4, pH 8.0. The resulting preparation was polished on a HiLoad 16/600 Superdex 200 pg column (Cytiva) using PBS. After elution, endotoxin was removed employing Polymyxin B agarose (Sigma Aldrich).
7
Egg Antigen Extraction and Purification
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SEA were prepared as previously described [16 (link),17 (link)]. Antigens were extracted from isolated eggs using sonication. The supernatant was recovered by centrifugation and filter-sterilized with a 0.22 μm filter and the endotoxin was removed with Polymyxin B-Agarose (Sigma-Aldrich). The remaining endotoxin activity (<0.01 EU/μg) was determined using the LAL assay kit (ThermoFisher Scientific, MA, USA). Protein concentration was determined using the DC Protein Assay Kit (Bio-Rad, Hercules, CA).
8
TLR4 and MyD88 Signaling Pathway Assay
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TLR4−/−, myD88−/−, Tirap/Mal,−/− Trif−/−, and female Balb/c mice at the age of 3–4 weeks old were purchased from Charles River Laboratories, Inc. (Wilmington, MA). The isolated monocytes were seeded in 96-well plates (100 cells/μl) in DMEM supplemented with GlutaMAX-I (2 mM), streptomycin (100 μg/ml), penicillin (100 U/ml), and heat inactivated 10% (v/v) FBS (Invitrogen, Waltham, MA). After the cells were stimulated for 24 h at 37°C, the supernatants were collected by centrifugation (400 × g, 4°C, 10 min). The LPS (E. coli serotype O55:B5) supplemented in cell culture and triacylated control lipopeptide, racemic Pam3, were purchased from Sigma-Aldrich (St. Louis, MO). Affinity chromatography on polymyxin B agarose (Sigma-Aldrich, St. Louis, MO) was used to remove LPS from recombinant proteins.
9
Recombinant Rv3628 Protein Production
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To produce recombinant Rv3628 protein, the corresponding gene was amplified by PCR using Mtb H37Rv ATCC 27294 genomic DNA as a template and the primer sequences 5′-CAATTCGACGTGACCATCGAA-3′ and 3′-GTGTGTACCGGCCTTGAAGCG-5′. The rv3628 gene was inserted into a pET22b (+) plasmid (Novagen, Madison, WI), and the resultant products were sequenced. Plasmids containing recombinant Rv3628 were transformed into E. coli BL21 cells by heat-shock for 1 min at 42°C. After cell disruption via sonication, recombinant Rv3628 was purified using Ni-NTA resin as previously described with slight modifications [8 (link)]. To remove endotoxins, the dialyzed recombinant protein was incubated with polymyxin B-agarose (Sigma, St. Louis, MO, USA) for 6 h at 4°C. Finally, the purified endotoxin-free recombinant protein was filter-sterilized and frozen at -70°C. The protein concentration was estimated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard.
10
Cloning and Purification of Rv3131
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Rv3131 was cloned as described previously with slight modifications27 (link). Briefly, Rv3131 gene was amplified (supplementary information: Figure S1 ) using H37Rv DNA as a template and cloned into pET28a plasmid between BamHI and Hind III restriction enzyme sites. The plasmid was then transformed in to E.coli BL21 (DE3) and the recombinant protein was induced with 0.5 mM IPTG (Sigma Aldrich, USA) for 4 hours in LB broth containing Kanamycin 50 μg/ml. Protein extraction was carried out as described earlier27 (link). The purity of recombinant Rv3131 (rRv3131) was checked by electrophoresing different fractions of the eluates on SDS gels. Rv3131 resolved as a single band and the purity of the protein as visualised was more than 95%. Endotoxin contamination was removed by incubating the protein with polymyxin-B agarose (Sigma, MO, USA). Limulus amebocyte lysate assay (Pierce™ LAL Chromogenic Endotoxin Quantitation Kit, ThermoFisher, USA) was used to measure the endotoxin content of the recombinant protein. Endotoxin contamination was undetectable in the protein fractions incubated with polymyxin-B agarose.
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