Factor Xa Cleavage of GST from Fused protein bound to Glutathione-Agarose

FACTOR Xa CLEAVAGE OF GST FROM FUSED PROTEIN BOUND TO GLUTATHIONE-AGAROSE
	Last Update: December 2006
	References: Nagai K, Thrgersen H (1984) Nature 309: 810; Nagai K, Thrgersen H (1987) Methods in Enzymol 153: 461

PREPARE SOLUTIONS
1. Factor-Xa buffer (40 mL):	Mix 0.8 mL of 100mM CaCl₂ (2mM), 0.8 mL of 10M NaCl (200mM), 4 mL of 1M Tris-HCl pH 8.0 (100mM), and add dH₂O to 40 mL

PROCEDURE
NOTE: The first AA after the Factor Xa recognition site (Ile-Glu-Gly-Arg-X) is Arg. Make sure that a similar sequence is not found at the fusion protein, and no R or P amino acid after this site
1. Follow protocol of loading and washing GST-fusion protein onto a 2 mL slurry of glutathione-agarose beads
2. After the last wash with PBS; EDTA, wash the resin still bound to GST-fusion with ~10 mL Factor-Xa buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 1mM CaCl₂)
3. Mix for 10 min, centrifuge at 500x g for 2 min, discard supernatant. Repeat the wash again and after 5 min centrifugation discard supernatant and leave the pellet as dry as possible
4. Add fresh 2 mL of Factor Xa buffer to the resin, and Factor Xa (1-10 mg, if you start with 50 mL bacteria culture) (BMB ). Incubate for 2-16 h at 20^oC while mixing
NOTE: In some cases addition of 0.3% SDS to the reaction improve cleavage
One unit will cleave >90% of 100 mg of a GST-protein in 50mM Tris-HCl, pH 7.5; 150mM NaCl; 1mM CaCl₂ at 22^oC for 16 hours
For each mL of glutathione Sepharose bed volume, mix 50 mL of resuspended Factor Xa (1U/mL) and 950 mL of 1X PBS or 50mM Tris-HCl, pH 7.5; 150mM NaCl; 1mM CaCl₂
5. Centrifuge for 5 min at 500 xg, collect the eluted protein, that also contain Factor-Xa (add 2 mL of 0.1M PMSF to inactivate the protease). Add to the beads 1 mL more of Factor-Xa buffer mix 10 min, centrifuge and save eluted protein. Repeat again and save separately each of the 3 eluents
6. Elute the GST-protein from the agarose beads by adding 5 mL of elution buffer, mix at room temperature for 20 min, centrifuge for 5 min at 500 g, collect the eluted protein. Add to the beads 5 ml more of elution buffer, mix 10 min, centrifuge and save eluted protein. Repeat again and save separately each of the 3 eluents
7. Column can be regenerated