Superdex 200 increase column

NKA was isolated from rectal glands of shark Squalus acanthias as described previously (11 (link)). The microsomal preparation was purified by sucrose flotation (27 (link)) and washing with ∼0.15% deoxycholate. The preparation was suspended in histidine/ethylenediaminetetraacetic acid (EDTA) buffer with 25% glycerol and kept at −80 °C until use. This preparation contained the α₁- and β₁-subunits and an equimolar amount of the FXYD10 regulatory protein (28 (link)) and showed a turnover number of 200/s at 37 °C.
For solubilized NKA, the purified membrane preparation was incubated first with 10 mM 3-morpholinopropanesulfonic acid (MOPS)–n-methyl-D-glucamine (NMDG), pH 6.5; 25% (wt/vol) glycerol; 4 mM MgCl₂; and 4 mM glutathione. It was then treated with 5.2% (wt/vol) octaethyleneglycol mono-n-dodecylether (C₁₂E₈) at a weight ratio (C₁₂E₈/protein) of 3.5 and separated from the insoluble fraction by centrifugation at 200,000 × g at 10 °C. The supernatant was subjected to size-exclusion chromatography using a Superdex 200 Increase column (Cytiva) equilibrated with 1 mM MgCl₂, 0.01% (wt/vol) C₁₂E₈, 1 mM dithiothreitol, and 20 mM 2-morpholinoethanesulfonic acid–NMDG, pH 6.1, or 20 mM imidazole-HCl, pH 7.5. Peak fractions were collected and concentrated to 8 mg/mL and stored in liquid nitrogen until use.

NEU1 and CTSA were expressed as secreted proteins in Sf9 insect cells infected with recombinant baculovirus. For murine NEU1 (UniProt ID O35657), the endogenous 41-residue N-terminal signal peptide (SP) was replaced by the melittin SP MKFLVNVALVFMVVYISYIYA, followed by a hexahistidine tag DRHHHHHHGS. The 47-residue SP of human NEU1 (UniProt ID Q99519) and the 28-residue SP of human CTSA (UniProt ID P10619) were also replaced as above. The genes were cloned downstream of a polyhedrin promoter.
Expression was carried out at 27°C for 66 hours in I-Max medium (Wisent). Proteins were isolated from expression culture media by immobilized metal affinity chromatography with HisPur Ni-NTA resin (Thermo Fisher Scientific), washed with buffer [25 mM tris-HCl (pH 7.5), 500 mM NaCl, and 10 mM imidazole], and eluted with buffer containing 250 mM imidazole. Proteins were purified by size exclusion chromatography (SEC) on a Superdex 200 Increase column (Cytiva) in buffer [10 mM tris-HCl (pH 7.5) and 100 mM NaCl]. NEU1 was further purified by anion exchange chromatography on a Mono Q column (Cytiva) in buffer [10 mM tris-HCl (pH 7.5) and 100 to 300 mM NaCl], diluted to 100 mM NaCl, and concentrated to 10 mg/ml. CTSA was supplemented with 10 mM sodium acetate (pH 4.5) and concentrated to 20 mg/ml, as it underwent reversible aggregation at high concentration at neutral pH.