Hiprep 26 10 desalting

The solid support was reacted with MeNH₂ in EtOH (33%, 0.5 mL) and MeNH₂ in water (40%, 0.5 mL) for 7 h at room temperature. The supernatant was removed from and the solid support was washed 3× with THF/water (1/1, v/v). The combined supernatant and the washings were evaporated to dryness. The resulting residue was treated with tetrabutylammonium fluoride trihydrate (TBAF·3H₂O) in THF (1 M, 1 mL) at 37 °C overnight to remove the 2’-O-silyl protecting groups. The reaction was stopped by the addition of triethylammonium acetate (TEAA) (1 M, pH 7.4, 1 mL). The volume of the solution was reduced and the solution was desalted using a size exclusion column (GE Healthcare, HiPrep 26/10 Desalting; 2.6 × 10 cm; Sephadex G25) eluting with H₂O, the collected fraction was evaporated to dryness and dissolved in 1 ml H₂O. The crude RNA after deprotection was analyzed by anion-exchange chromatography on a Dionex DNAPac PA-100 column (4 mm × 250 mm) at 80 °C. Flow rate: 1 mL min^-1, eluant A: 25 mM Tris·HCl (pH 8.0), 6 M urea; eluant B: 25 mM Tris·HCl (pH 8.0), 0.5 M NaClO₄, 6 M urea; gradient: 0–60% B in A within 45 min, UV detection at 260 nm.

Fractions eluted from IMAC were loaded onto a desalting column (HiPrep 26/10 desalting, GE Healthcare, Milan, Italy). In order to reduce salt concentration and remove imidazole, proteins were eluted with ultrapure water (MilliQ; Millipore, USA) in 1.5 column volumes (CV) at a flow rate of 1 ml/min. Eluted proteins were detected at 214 and 280 nm and then separated by reverse phase chromatography (RPC).

To examine the interaction between AtxA and DNA sequences in vitro, the atxA gene was cloned into the expression vector pET 28-b (Novagen, Madison, WI, USA) with N- and C-terminal 6xHIS-tags and an internal FLAG-tag. The BL21 (DE3) expression strain of E. coli was then transformed with an AtxA expression vector. Cells harboring the AtxA expression vector were cultured at 37 °C in LB medium containing 50 μg/ml kanamycin. After overnight growth, cells were diluted to an OD600 of 0.05 in 200 ml of LB medium containing kanamycin and cultured to an OD600 of 0.5. Next, the cells were induced by adding one mM IPTG and cultured at 25 °C for 16 h. After incubation, the cells were collected by centrifugation at 10,000×g for 10 min and stored at −80 °C. Thawed cells were suspended in eight ml binding buffer (50 mM NaH₂PO₄, 300 mM NaCl, 0.5% Triton X-100, 10 mM imidazole, one mM PMSF, pH 8.0) and disrupted by sonication. After centrifugation for 15 min at 15,000×g, recombinant AtxA protein was purified by Ni-NTA (Qiagen, Hilden, Germany). The extract was then desalted by HiPrep 26/10 Desalting (GE, Boston, MA, USA) and purified by RESOURCE S, one ml (GE, Boston, MA, USA) using 25 mM MES, pH 6.5. The final concentration of recombinant AtxA was determined by a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).

For antibody purification, L2A5 hybridoma cells were cultured in serum-free medium (Hybridoma-SFM, Thermo Scientific, 12045076). Hybridoma supernatant containing L2A5 mAbs was recovered and centrifuged at 1000 g for 10 min. The supernatant was collected and diluted twofold in PBS. Considering the composition of the Hybridoma-SFM medium used, low level of contaminants was present in the recovered supernatant and therefore a desalting strategy was considered useful to recover and purify the anti-STn IgM antibodies. For that, the supernatant was loaded onto desalting columns (HiPrep 26/10 Desalting, GE Healthcare Life Sciences, 45-000-266) and dialyzed against PBS (pH 7.2). Due to the high volume of supernatant available, four desalting columns were connected and used in series. The protein peak was registered by spectrophotometry measuring the OD at 280 nm. Eluted fractions containing antibodies were further concentrated using a 200 kDa MW cut-off concentrator system (Vivaflow® 200, Sartorius). A final concentration step to reduce the final sample volume was performed by ultrafiltration using a 100 kDa MW cut-off filter (Amicon, Millipore). Antibody concentration was assessed using the Pierce™ modified Lowry protein assay kit (Thermo Scientific, 23240)⁵⁹ .

All chemicals and media were from Sigma-Aldrich (ST.Louis, USA), unless indicated otherwise. Bicinchoninic acid (BCA) assay reagent was from Pierce (Rockford, IL, U.S.A.). DNAse I was from Roche Diagnostics (Mannheim, Germany) and chicken egg white lysozyme was obtained from Fluka (Buchs, Switzerland; 84,468 U/mg). Hiprep 26/10 desalting and Superdex Peptide 10/300 columns were purchased from GE Healthcare, (Uppsala, Sweden). PageRuler™ Prestained Protein Ladder was from (Fermentas, Vilnius, Lithuania). Phosphate buffer saline (PBS) was obtained from Braun (Melsungen AG, Germany).

The expression vector, pBsHSD-26b, was transformed into Rosetta2 (DE3) strain of E. coli for overexpression. The cells were cultured in LB media at 37°C with shaking until the OD₆₀₀ reached 0.5-0.8. Then, the culturing flask was cooled in an ice bath for about 10 min and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce BsHSD expression. The cells were cultured again for an additional 20 h at 25°C. The cultured cells were harvested by centrifugation at 1,590 g for 20 min (Hanil Supra 22K), and then the cell pellets were resuspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 0.4 M NaCl, 0.1 mM TCEP, 5%glycerol, 10 mM imidazole and 0.1 mM phenyl methyl sulfonyl fluoride (PMSF). The resuspended cells were lysed by sonification and then centrifuged at 24,650 g for 60 min (Hanil Supra 22K). Next, the BsHSD was purified from the supernatants through 3 serial applications of column chromatography using a HisTrapFF, HiPrep 26/10 Desalting and Superdex-200 (GE Healthcare, USA). The protein purity was examined with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and the concentration was measured using a NanoDrop 1000 (Thermo Scientific, USA).

N-Formyl-l-methionine pentafluorophenylester
was synthesized as described
in references (32 (link)).
One equivalent of 3′-amino-3′-deoxyoligoribonucleotides 4 (final concentration = 0.1 mM) and 200 equiv of N-formyl-l-methionine pentafluorophenylester (final
concentration = 20 mM) were dissolved in 100 mM Tris–HCl (pH
8.0) and dimethyl sulfoxide (1/1, v/v). The typical total reaction
volume amounted to 150 μL. After 15 min at 37 °C, the reaction
mixture was diluted with 450 μL water and directly applied on
a size-exclusion chromatography column (GE Healthcare, HiPrep 26/10
Desalting, 2.6 × 10 cm, Sephadex G25). By elution with H₂O, the conjugate-containing fractions were collected and evaporated
to dryness, and the residue was dissolved in H₂O (1 mL).
Analysis of the crude products was performed by anion-exchange chromatography
on a Dionex DNAPac PA-100 column (4 × 250 mm) at 60 °C (flow
rate: 1 mL min^–1; eluent A, 25 mM Tris-HCl (pH 8.0)
and 20 mM NaClO₄ in 20% aqueous acetonitrile; eluent B,
25 mM Tris-HCl (pH 8.0) and 0.60 M NaClO₄ in 20% aqueous
acetonitrile; gradient: 0–35% B in A within 30 min; UV detection
at λ = 260 nm).