Endotoxin e coli standards kits

A 1065 bp fragment of cDNA encoding Sjp40 and a 780 bp fragment of cDNA encoding Sjp90α were amplified by PCR and inserted into the pET28b vector (Invitrogen, Carlsbad, CA, USA). E. coli BL21 (DE3) cells (Invitrogen) were transformed with the reconstructed plasmids for protein expression as described [51 (link)]. The expressed rSjp40 and rSjp90α proteins were purified from E. coli lysates using Ni-NTA affinity chromatography (GE Health Life Science, Pittsburgh, PA, USA). Given previous studies showing the 6 × His tag did not stimulate any immunogenic activity [52 (link),53 (link),54 (link),55 (link)], the tag was not removed after purification of rSjp40 and rSjp90α. However residual endotoxin was removed from both recombinant proteins as described [51 (link)] and Endotoxin (E. coli) Standards kits (Lonza, Basel, Switzerland) were used subsequently to confirm there was no Endotoxin contamination in either protein.

A 1065 bp fragment of cDNA encoding SjLD1 [16 (link)] and a 714 bp fragment of cDNA encoding SjTPI [37 (link)], were amplified by PCR and inserted into the pET28b vector (Invitrogen, Carlsbad, CA, USA), respectively. E. coli BL21 (DE3) cells (Invitrogen) were transformed with the reconstructed plasmids for protein expression as described [9 (link)]. The expressed SjTPI and SjLD1 proteins were purified from E. coli lysates using Ni-NTA affinity chromatography (GE Health Life Science, Pittsburgh, PA, USA). Recombinant SjLD1 (rSjLD1) protein was further purified (denaturing conditions; 6 M guanidine-HCl) and refolded in buffer (300 mM NaCl, 50 mM NaH₂PO₄, 8% w/v sucrose, PH 4.5). Purified proteins had residual endotoxin removed [9 (link)] and Endotoxin (E. coli) Standards kits (Lonza, Basel, Switzerland) were used subsequently to assess any contamination. The purified rSjLD1 and rSjTPI proteins were used in animal vaccine/challenge experiments and for immunological analysis.