Cloning, expression and purification of recombinant Sm-p80

Recombinant Sm-p80 (rSm-p80) was produced using prokaryotic expression system. Briefly, the full length coding sequence of Sm-p80 gene sequence (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M74233","term_id":"160934","term_text":"M74233"}}M74233) was cloned into pCold II vector (GenScript Corp., Piscataway, NJ, USA). The recombinant plasmid was transformed into BL21 (DE3) Escherichia coli strain. Production of recombinant protein was induced with 0.75 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at mid-log phase (OD₆₀₀ ~ 0.6 – 0.8) at 15°C and bacterial cells were harvested 24 h post-induction. Harvested cells were lysed with denaturing lysis buffer (49.9 mM NaH₂PO₄, 299 mM NaCl, 8 M Urea and 19 mM Imidazole, pH 8.0) followed by sonication. Cell lysate was then centrifuged at 18,000 × g for 30 min at 4°C. The supernatant was collected and centrifuged again as above to remove remaining cellular debris. The expressed protein was purified by utilizing Immobilized metal affinity chromatography (IMAC) (Bio-Rad, CA, USA) followed by size exclusion chromatography using Sephadex G-150 columns. The purified denatured protein was refolded through step-wise dialysis in several exchanges of dialysis buffer (20 mM Tris, 150 mM NaCl, 5% Glycerol, pH- 8.0) and the protein concentrated using Spin-X UF concentrator (Corning, MA USA). Endotoxin levels in protein samples were analyzed with a Limulus amebocyte lysate assay (Charles River Laboratories International, Inc., Wilmington, MA, USA). The quality of recombinant Sm-p80 produced was analyzed by Sodium Dodecyl Sulfate Polyacrylamide gel (SDS PAGE) and western blotting using Horseradish peroxidase (HRP)-conjugated antibody directed against the recombinant His-tag (Thermo Fisher Scientific, MA, USA) (Online Resource 1). Endotoxin levels in the recombinant Sm-p80 used in immunizations were below that approved by the Food and Drug Administration for human use (approximately 0.06 EU/ml).