Endotoxin removal kit

The pET30a vector (constructed in our laboratory) was used to express N. farcinica Nfa34810 in E. coli. The suicide plasmid pk18mobsacB (constructed in our laboratory) was used to construct the nfa34810 deletion mutant. The following antibodies were used in this study: anti-p-Jnk and -JNK (4668; Cell Signaling), anti-p-ERK 1/2 and -ERK 1/2 (4370; Cell Signaling), anti-p-p38 and -p38 (4511; Cell Signaling), anti-p-p65 and -p65 (3033; Cell Signaling), anti-p-AKT and -AKT (4060; Cell Signaling), anti-β-actin (TransGen Biotech), anti-TLR4 (NB100-56727; Novus Biological, USA), anti-TLR2 (NB100-56726; Novus Biological, USA), and IgG_2A isotype control (MAB003; R&D Systems, USA). Human and mouse TNF ELISA kits were used in this study (BD, USA). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059), JNK inhibitor (SP600125), and NF-κB inhibitor (BAY 11-7082). An endotoxin removal kit was purchased from GenScript (Nanjing, China).

PCR amplified the Fn-Dps gene using primers F1 and R1 (S1 Table) and Fn ATCC 25586 DNA as templates. The PCR products were digested with Nco I and an Xho I and ligated into pET28a (+) expression vector (Novagen, USA). The recombinant plasmids of pET28a-Fn-Dps were transformed into E. coli BL21 (DE3). The expression of the recombinant Fn-Dps was induced with 0.8 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for continuous 16 h cultivation at 25°C. The recombinant Fn-Dps with a His tag was purified using a Ni-NTA column. (Sangon Biotech, China). The endotoxin in Fn-Dps was removed using the Endotoxin Removal Kit (GenScript, China).
The total bacterial proteins from IPTG-induced E. coli BL21 harboring pET28a (+) empty vector were removed endotoxin and then quantified and used as a control protein. The protein concentration was determined with a BCA Kit (Beyotime, China).

Plasmid construct (pRK172-human-α-synuclein) encoding wild-type human α-synuclein was expressed in BL21-CodonPlus (DE3-RIL) cells (Agilent, catalog 230245-41). Protein expression was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside at cell density OD (600 nm) 0.8 and overnight incubation at 18°C with continuous shaking. Cell pellets were lysed in 0.75 M NaCl, 10 mM Tris HCl pH 7.6, 1 mM EDTA, and 1 mM PMSF and sonicated at 30% power (Thermo Fisher Scientific 500 Dismembrator) for 1 minute followed by boiling the cell suspension 15 minutes. Centrifuged and filtered samples were dialyzed against 10 mM Tris HCl, pH 7.6, with 50 mM NaCl, 1 mM EDTA, and 1 mM PMSF. The suspension was passed through a HiPrep Q HP 16/10 column, 1 × 20 mL, on an ÄKTA pure protein purification system (both Cytiva, formerly GE Healthcare) with running buffer composed of 10 mM Tris pH 7.6 and 25 mM NaCl, then eluted with a linear gradient application of high-salt buffer (10 mM Tris HCl, pH 7.6, 1 M NaCl). Fractions containing a single band of α-synuclein were identified by Coomassie staining of SDS-PAGE gels and were dialyzed and concentrated. Purified monomer protein was subjected to 2 to 3 rounds of endotoxin removal (Endotoxin removal kit, GenScript) until a level of < 0.1 EU/mg was achieved. Endotoxin levels were determined using an LAL chromogenic endotoxin quantification kit (GenScript).