Polymyxin b agarose column

Depletion of LPS was performed using a Polymyxin B-Agarose column (Sigma-Aldrich). The column was washed with endotoxin-free 0.1 M ammonium bicarbonate buffer (pH = 8.0), in order to remove the glycerol storage solution; then it was centrifuged at 9,600 g for 5 min and resuspended with an equal volume of BE. The polymyxin B-Agarose-BE suspension was stirred for 30 min at 37°C, and then it was centrifuged at 9,600 g for 10 min and the supernatant (BE-LPS) was reserved.
LPS was eluted from the column using 1% sodium deoxycholate (Sigma-Aldrich) and then the solution was dialyzed to eliminate the salt and resuspended in endotoxin-free water.

The r-LdODC protein was cloned, expressed, and purified according to the previous study [26 (link)]. In the recombinant protein, Bacterial lipopolysaccharide/endotoxin (LPS) impurity was checked by using the Limulus amoebocyte lysate (LAL) test (Thermo Fisher Scientific Nunc, MA, USA). Endotoxin was removed by passing r-LdODC protein through a polymyxin B-agarose column (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Endotoxin-free protein was used in further study.
Apart from recombinant protein, the five most potent immunogenic LdODC-derived HLA-DRB10201 (MHC Class-II) restricted 15 mer epitopes against Homo sapiens namely, YNVVTRLPASPAALA (P1), SERIRMAPPASASKA (P2), PGRYFTAASHALLMN (P3), ALLMNVFASRTLRLS (P4), and EKISRLMPSAHAIIR (P5) were identified by SYFPEITHI, IEDB, and NetMHCCII-2.2 bioinformatic tools. Further population coverage and antigen cross-presentation were analyzed and screened by the IEDB population coverage analysis tool and NetMHCpan 2.3, respectively. Additionally, a 100 ns molecular dynamics simulation study was performed for each selected epitope at 300 K. The shortlisted epitopes were synthesized by Peptide2.0 (Chantilly, VA, USA) with 95% purity [16 (link)]. The purified protein and synthesized peptides were stored at −80 °C for further in vivo study.