125
2. Follow the steps 2–5 in Subheading 3.2.
3. Amplify the DNA fragment using primers FlucEcoBack and
FlucPacR437KF or using the plasmid constructs generated in
steps 1 and 2 as the template.
4. Digest the plasmid constructs generated in steps 1 and 2 with
EcoRI and PacI.
5. Recombine the fragments of steps 3 and 4 using an In-Fusion
HD cloning kit as per manufacturer’s instructions.
1. Transform E. coli SHuffle T7 Express lysY competent cells
with pET32/BD1/Donor and pET32/BD2/Acceptor (see
Note 9).
2. Incubate the transformed cells on a LBA agar plate at 30 °C
for 24 h.
3. Pick a colony, and culture it in 4 mL LBA medium at 30 °C.
4. Centrifuge 0.4 mL of the resultant cell culture and collect the
cell pellet (see Note 10).
5. Resuspend the cell pellet with 100 mL of LBA medium in a
500 mL flask, and culture at 30 °C.
6. When the OD 600 reaches 0.4–0.6, add IPTG to a final concentration of 40 μM and culture the cells at 16 °C.
7. Centrifuge and collect the cells, and resuspend the cell pellet
with 10 mL of the TALON extraction buffer.
8. Sonicate the cells on ice using an ultrasonifier for 2 min (50%
interval) five times (see Note 11).
9. Centrifuge the sonicated solution at 11000 × g for 20 min at
4 °C.
10. Add 200 μL of TALON Metal Affinity Resin to the supernatant, and rotate or shake gently for 20 min at 25 °C.
11. Centrifuge at 700 × g for 2 min at 4 °C, and resuspend the
resin with 10 mL of TALON extraction buffer.
12. Rotate or shake gently for 10 min at 4 °C.
13. Repeat steps 11 and 12 two times.
14. Centrifuge at 700 × g for 2 min and collect the resin.
15. Suspend the resin with 3 mL of TALON extraction buffer, and
transfer the suspension into a TALON 2 mL Disposable
Gravity Column.
16. Allow to drain until it reaches the top of the resin bed.
17. Wash the column with 2 mL of TALON extraction buffer.
18. Elute the enzyme with 500 μL of TALON elution buffer (see
Note 12).
3.4 Expression
and Purification
of the BD1/Donor
and the BD2/Acceptor
Sensitive Protein-Protein Interaction Assay
2. Follow the steps 2–5 in Subheading 3.2.
3. Amplify the DNA fragment using primers FlucEcoBack and
FlucPacR437KF or using the plasmid constructs generated in
steps 1 and 2 as the template.
4. Digest the plasmid constructs generated in steps 1 and 2 with
EcoRI and PacI.
5. Recombine the fragments of steps 3 and 4 using an In-Fusion
HD cloning kit as per manufacturer’s instructions.
1. Transform E. coli SHuffle T7 Express lysY competent cells
with pET32/BD1/Donor and pET32/BD2/Acceptor (see
Note 9).
2. Incubate the transformed cells on a LBA agar plate at 30 °C
for 24 h.
3. Pick a colony, and culture it in 4 mL LBA medium at 30 °C.
4. Centrifuge 0.4 mL of the resultant cell culture and collect the
cell pellet (see Note 10).
5. Resuspend the cell pellet with 100 mL of LBA medium in a
500 mL flask, and culture at 30 °C.
6. When the OD 600 reaches 0.4–0.6, add IPTG to a final concentration of 40 μM and culture the cells at 16 °C.
7. Centrifuge and collect the cells, and resuspend the cell pellet
with 10 mL of the TALON extraction buffer.
8. Sonicate the cells on ice using an ultrasonifier for 2 min (50%
interval) five times (see Note 11).
9. Centrifuge the sonicated solution at 11000 × g for 20 min at
4 °C.
10. Add 200 μL of TALON Metal Affinity Resin to the supernatant, and rotate or shake gently for 20 min at 25 °C.
11. Centrifuge at 700 × g for 2 min at 4 °C, and resuspend the
resin with 10 mL of TALON extraction buffer.
12. Rotate or shake gently for 10 min at 4 °C.
13. Repeat steps 11 and 12 two times.
14. Centrifuge at 700 × g for 2 min and collect the resin.
15. Suspend the resin with 3 mL of TALON extraction buffer, and
transfer the suspension into a TALON 2 mL Disposable
Gravity Column.
16. Allow to drain until it reaches the top of the resin bed.
17. Wash the column with 2 mL of TALON extraction buffer.
18. Elute the enzyme with 500 μL of TALON elution buffer (see
Note 12).
3.4 Expression
and Purification
of the BD1/Donor
and the BD2/Acceptor
Sensitive Protein-Protein Interaction Assay