Histrap ff 1 ml column

Transformed E. coli strains were grown to an OD₆₀₀ of 0.5 in LB medium containing the appropriate antibiotics at 37 °C and 250 rpm. IPTG at a final concentration of 0.1 mM was added, and the cultures were incubated overnight to induce protein production at 12 °C and 250 rpm. Cells were harvested using centrifugation at 11,000 rpm and 4 °C for 15 min, and the resulting pellets were resuspended in 30 mL of BugBuster Protein Extraction Reagent containing Benzonase Nuclease (Merc Millipore, Darmstadt, Germany). After incubation at 4 °C for 30 min, centrifugation at 11,000× g and 4 °C for 20 min was performed to remove cell debris. The supernatant was filtered through 45 µm syringe filters (Whatman, GE Healthcare Life Sciences, Pittsburgh, PA, USA) and applied to a 1 mL HisTrap FF column (GE Healthcare Life Sciences, Pittsburgh, PA, USA) at a flow rate of 1.0 mL min⁻¹. The bound proteins were eluted with 20 mM HEPES, 400 mM imidazole, and 400 mM NaCl (pH 7.5) after washing with 20 mM HEPES, 20 mM imidazole, and 400 mM NaCl (pH 7.5). Fractions corresponding to the absorbance peak at 280 nm were collected and verified using SDS-PAGE and then pooled and desalted with 20 mM HEPES (pH 7.0) using an Amicon Ultra 15 mL Filter (Merck Millipore, Darmstadt, Germany) with an MW cutoff of 10 kDa. All purification steps were performed at 4 °C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

The gene for toxin-simulating protein (TSP) was designed to include beta-galactosidase from E. coli BL21 on its N-terminus (gene bank code CAQ30819) and the receptor binding domain of BoNT/A, also designated H_C fragment, on its C-terminus (gene bank code M30196, amino acids 872-1296). The two proteins were connected by a flexible linker with the sequence (GGGGS)₃ and a His6 tag was added to the C-terminus. The receptor-simulating protein (RSP) gene consists of a GST tag on its N-terminus and the fourth luminal domain of Mus musculus SV2C (amino acids 545–580, GenBank code AAI37862.1) on its C-terminus. Synthetic genes with optimized codon usage were prepared by GenScript (Piscataway, NJ, USA) and cloned into pET-9a.
To produce proteins, E. coli BL21(DE3) harboring pET-9a-TSP or pET-9a-RSP was grown in TB media at 18 °C and 250 rpm for ~40 h, and thereafter, the cells were harvested by centrifugation and disrupted by sonication. TSP and RSP were purified using a HisTrap FF 1 mL column and a GSTrap FF 5 mL column (GE Healthcare), respectively, according to the manufacturer’s instructions.

E. coli BL-21 (DE3) strain carrying pOG-His-PhoP was grown overnight in LB medium containing 50 µg/ml Kan at 37°C with vigorous aeration. The culture was diluted 1∶200 into 300 ml of fresh LB and grown for 2.5 h. To induce expression of the recombinant protein, IPTG was added to 1 mM final concentration, and the culture was grown for an additional 3 h at 30°C and harvested by centrifugation. The pellet was resuspended in 10 ml lysis buffer [50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 1 mM MgCl₂ in the presence of DNase I (5 µg/ml) and RNase A (10 µg/ml)]. Cells were disrupted using a French Press (three passages, 1000 PSI) and then centrifuged at 20,000 rpm for 30 min. His₆-PhoP was purified from 1 ml of the soluble fraction by a nickel-affinity chromatography using ÄKTApurifier FPLC system (GE Healthcare) and a HisTrap FF 1 ml column (GE Healthcare) at 4°C. The column was washed with 5 ml of washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole and 1 mM MgCl₂). His₆-PhoP was eluted with 5 ml elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole and 1 mM MgCl₂) and analyzed on an SDS-polyacrylamide gel. The selected elute fractions were then collected and dialyzed against dialysis buffer [20 mM HEPES (pH 8.0), 100 mM KCl, 20% glycerol, 1 mM DTT and 1 mM MgCl₂] on a Sephacryl S-200 gel filtration column. Dialyzed fractions were analyzed by UV absorbance at 280 nm and on an SDS-polyacrylamide gel. The appropriate fractions were pooled and concentrated using a 10 kDa Amicon Ultra centrifugal filter devices (Millipore). Protein samples were stored at −80°C until use.