Recombinant S protein purification by IMAC
In a typical process, 75 g of infected larvae were homogenized directly
with 200 ml of equilibration buffer containing 10 mg glutathione
crystals, 50 mM arginine, and 4 mM PMSF and 1/200 (V/V) protease
inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA) using a Bag
Mixer 400 homogenizer (Interscience, Saint-Nom-la-Bretèche, France).
Then, the larval extract was centrifuged at 10,000×g for 30 min at 4 °C
and the pellet was discarded. The supernatant was filtered through
Whatman paper using a filter holder with a receptor (Nalgene, USA) to
remove the lipid fraction remaining at the top. The filtered sample was
centrifuged for a second time in the same conditions, and the
supernatant was filtered through 3 µm (Sartopore o SartoBran capsules).
Then two experiments were conducted. In the first experiment, the
clarified homogenate was loaded into the HisTrap FF 5 ml column (Cytiva,
Marlborough, USA) previously equilibrated with 10CV (column volume) of
20 mM phosphate buffer, pH 7.4, 20 mM imidazole, 300 mM NaCl, and 50 mM
arginine, and washed with 10CV of the same buffer. The recombinant
protein was eluted with a buffer containing 500 mM imidazole, 100 mM
arginine, and 10% glycerol. In the second experiment, the same
chromatography was done but the matrix was equilibrated with the same
buffer containing 20 mM imidazole, a second wash was done with 80 mM
imidazole, and then the S protein was eluted with 500 mM imidazole.
After that, an optimized protocol consisted in equilibrating the
chromatographic matrix directly with the same buffer containing 80 mM
imidazole, and after washing, the S protein was eluted by adding 500 mM
imidazole. In all cases, the linear flow rate was 0.4 cm min-1, and all
fractions were collected and subjected to SDS-PAGE and Western blot
analysis.