TraY Purification

TraY Overexpression

1. Inoculate 500mL of LB+amp with a 500μL BL21(DE3) pET21a(+)+TraY starter culture.

2. Grow to OD₆₀₀=0.5 and induce by adding IPTG to a final concentration of 1mM; induce for 3-4hrs.

3. Spin down cells in 500mL sterilized bottle for 30minutes at 3700rpm or 20 minutes at 10,000 x g at 4˚C.

4. Remove supernatant and store pellet at -80˚C.

TraY Purification

5. Resuspend 500mL pellet in 50ml of ice-cold buffer (50mM Tris-HCl pH 7.5, 1mM EDTA, 5mM βME and 100mM NaCl).

6. Add PMSF to a final concentration of 200μM.

7. Sonicate cells at max voltage (14-16watts on Fambrough sonicator) for 1 minute bursts 6-8X (until lysate is clear). Between each burst allow cells to chill on ice for 2 minutes.

8. Spin cells at 10,000 x g for 10 minutes at 4˚C.

9. Load supernatant onto 5ml HiTrap Q column and run a salt gradient from 100mM to 2M at 5ml/min for 100ml.

10. Collect 2ml fractions and run them along with supernatant and flow through (FT) on 10% acrylamide native gel.

11. Load FT from Q column onto 5ml HiTrap Heparin column and run a salt gradient from 100mM to 1M at 5ml/min for 100ml.

12. Collect 3mL fractions and run them along with supernatant and flow through (FT) on 10% acrylamide native gel.

13. Load fractions containing TraY from Heparin column onto 5ml HiTrap Blue column and run a salt gradient from 500mM to 3M at 5ml/min for 100ml.

14. Collect 3ml fractions and run them along with supernatant and flow through (FT) on 10% acrylamide native gel.

15. Add equal amount of 4M NaCl to fractions from Blue column to bring protein salt concentration between 2-3M.

16. Pour 1ml butyl-sepharose 4 Fast flow column (50% ethanol/50% water).

17. Add 4ml high salt buffer (20mM NaPO₄ pH 7.4, 1mM EDTA, 5mM βME, 3M NaCl) to equilibrate column. DON’T disrupt the column bed.

18. Load TraY onto the 0.5ml butyl-Sepharose 4 Fast Flow column.

19. To elute protein, add 2ml low salt buffer (20mM NaPO₄ pH 7.4, 1mM EDTA, 5mM βME, 100mM NaCl). Cap the column and let sit for 30 minutes at room temperature.

20. Add 4ml of the low salt buffer and collect all fractions.

21. Combine fractions containing pure TraY and dialyze in 3500MWCO tubing against low salt buffer.

22. Change dialysis buffer 3X, 8hr changes.

TraY Purification Buffers

Q column:

A B

50mM Tris-HCl pH 7.5 50mM Tris-HCl pH 7.5

1mM EDTA 1mM EDTA

5mM βME 5mM βME

100mM NaCl 2M NaCl

Heparin column:

A B

20mM NaPO₄ pH 7.4 20mM NaPO₄ pH 7.4

1mM EDTA 1mM EDTA

5mM βME 5mM βME

100mM NaCl 1M NaCl

Blue column:

A B

20mM NaPO₄ pH 7.4 20mM NaPO₄ pH 7.4

1mM EDTA 1mM EDTA

5mM βME 5mM βME

500mM NaCl 3M NaCl