Hiprep 26 10

The protocol followed is similar to the one described in 2.4.3, with the difference that ammonium sulfate was used for the precipitation of the ELPs during the ITC cycles and DMSO was employed to aid their resolubilization. In particular, the ELP-containing supernatant was supplemented with 10% v/v saturated ammonium sulfate solution to aggregate the ELPs at room temperature. The first hot spin was conducted by centrifugation at 10,000 rpm, 15 min, 25 °C, and the ELP pellets were resolubilized in DMSO after a freeze–thaw cycle. The mixture was diluted with cold 15 mM phosphate 15 mM NaCl buffer pH 7.4 (final content of DMSO 10% v/v) before proceeding to the first cold spin at 15,000 rpm, 10 min, 4 °C. The ITC cycles were repeated another 3 times without the use of DMSO. For each cold spin, the volume of buffer to resolubilize the ELPs was reduced to half, and thus for every hot spin the volume of ammonium sulfate was also reduced to half. The supernatant isolated after cold spin 4 was desalted on a HiPrep 26/10 (GE Healthcare Life Sciences) with an AKTA Explorer 10 (GE Healthcare Life Sciences) at 1 mL/min ultrapure water.

All TAL effectors with residues 231–720 were subcloned into pET21b vector (Novagen). All the mutated dHax3 (231–720)s were generated by site-directed mutagenesis. To obtain purified protein for crystallization, plasmid was transformated into E. coli BL21 (DE3) and induced by 0.2 mmol/L isopropyl -D-thiogalactoside (IPTG) when the cell density reached an OD₆₀₀ of 0.8. After growth at 22°C for 16 h, the cells were harvested, re-suspended in the buffer containing 25 mmol/L Tris-HCl pH 8.0, and 500 mmol/L NaCl, and disrupted using sonication. The recombinant proteins were purified sequentially through Ni²⁺-nitrilotriacetate affinity resin (Ni-NTA, Qiagen), heparin column (GE Healthcare) and desalting column (Hiprep 26/10, GE Healthcare).

Cellic CTec2 was diluted 1:5 with 25 mM sodium citrate buffer (pH 4.8), passed through a 0.2-μm polyether sulfone (PES) syringe filter, and desalted in 10 mL aliquots using two serial HiPrep 26/10 desalting columns (GE Life Sciences, Piscataway NJ) equilibrated in the same buffer. Protein-containing fractions were pooled and protein concentration determined using the bicinchoninic acid protein assay (Pierce Rockford, IL). Enzyme samples were desalted less than 2 days before use, with fresh material being generated for each experiment as desalted commercial enzymes tend to precipitate within a few days.

One milliliter of a liposomal suspension (CLp & and TrLp) was centrifuged at 10,000× g for 10 min to remove any insoluble material. The supernatant was loaded onto a Hiprep 26/10 desalting column (GE Life Sciences, Marlborough, MA, USA). The liposomes were eluted using phosphate buffered saline (PBS, pH 7.4) at 2 mL/min while monitoring the absorbance at 430 nm to detect curcumin. All curcumin-containing fractions were pooled. Each purified liposome suspension was filtered through a 0.45-μ filter before being analyzed by Dynamic light scattering (DLS). DLS was performed using a standard laser light scattering spectrometer (BI-200SM) equipped with a BI-9000 AT digital time correlator (Brookhaven Instruments Co, Holtsville, NY, USA) to measure the hydrodynamic diameter distributions.

CRM₁₉₇ was solubilized in MES 500 mM pH 6.0 (12 mg/mL); BMPH (42 mg/mL, molar ratio BMPH/COOH groups CRM₁₉₇ = 10.42) and EDAC (3 mg/mL, molar ratio EDAC/COOH groups CRM₁₉₇ = 1.15) were added. Mixture was stirred for 1 h at RT, and then purified by desalting against NaH₂PO₄ 100 mM EDTA 10 mM pH 7.0 on a HiPrep 26/10 desalting column 53 mL, prepacked with Sephadex G-25 Superfine (G-25 53 mL) [GE Healthcare].

The fully modified tc-oligonucleotides 5′-tc(^MeCT^MeCGG^MeCTTA^MeC)-3′ and 5′-tc(GTAAG^MeC^MeCGAG)-3′ were synthesized by Synthena AG (Switzerland). The natural RNA oligonucleotide 5′-r(GUAAGCCGAG)-3′ was synthesized on the 10 μmol scale on a Pharmacia LKB Gene Assembler Special DNA Synthesizer using standard phosphoramidite solid-phase methods. The natural DNA oligonucleotides 5′-d(CTCGGCTTAC)-3′ and 5′-d(GTAAGCCGAG)-3′ were purchased from Microsynth AG (Switzerland). All oligonucleotides were purified by ion exchange HPLC using a DNAPac PA100 22 × 250 mm semipreparative column (Dionex) applying the following eluents: A (25 mM Tris, pH 7.0), B (25 mM Tris, 1.25 M NaCl, pH 7.0). The gradient was programed as follows: 15% of eluent B in A for 13 min, then 15% to 35% of eluent B in A in 11 min, then 100% B for 6 min The oligodeoxynucleotides were desalted using HiPrep 26/10 (GE Healthcare), characterized by ESI-MS, and lyophilized. Oligonucleotide concentrations were determined using a NanoDrop ND-100 UV−vis spectrophotometer (NanoDrop Technologies, Inc.).

Lipopolysaccharide (O9:LPS) was from Sigma-Aldrich, UK (L2387, >99% free of protein) and Vi capsular polysaccharide was from Sanofi Pasteur, UK (Typhim Vi^®). Flagellin (H) was purified from a flagellin-deficient S. Enteritidis strain that expresses S. Typhi H:d from a plasmid (University of Maryland, USA) (14 (link), 20 (link)). Cytolethal distending toxin B (CdtB) and pilus control protein L (PilL) coding sequences lacking transmembrane domains were PCR-amplified from S. Typhi CT18 strain genomic DNA, cloned into plasmid pET28b(+) (Novagen, UK) and expressed in Escherichia coli BL21(DE3)pLysS (Promega, WI, USA). Recombinant His-tagged CdtB and PilL were purified by tagging with nickel-coated agarose beads (Ni-NTA, Invitrogen) and elution from gravity flow columns (Qiagen, Germany). CdtB was renatured in 50-mM sodium phosphate solution and 500-mM NaCl. hlyE was PCR-amplified from Ty21a, cloned into pET21a vector (Novagen, UK), and expressed in an LPS-modified E. coli BL21 strain to produce endotoxin free proteins (Lucigen, ClearCoil BL21 cells). Recombinant His-tagged hemolysin E (HlyE) was purified using HisTrap nickel-affinity column (GE Healthcare) followed by desalting on a HiPrep 26/10 (GE Healthcare).