Editing GST-3C Protease Purification

1. Freshly transform the 1613 pET28-cold-GST-3C construct into E. coli BL21 Star (DE3) pRARE2-pLysS. Plate out on LB-agar containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C. 

<br>2. Pick 3 colonies from the plate to inoculate 100 ml LB medium containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at<br>37°C in a shaker/incubator. 

<br>

3. Use the overnight cultures to inoculate 8 x 1 L LB medium containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml) in 2 L culture flasks. 

<br> 

4. Grow the cultures at 37°C until the OD600 is approx. 1 to 1.5. Then lower the temperature of the shaker/incubator to 20~25 °C, add 1 M IPTG to 0.1 M final conc, and incubate the cultures overnight. 

<br> 

5. Harvest the cells by spinning for 5 min at 6,000 rpm. Weigh pellet. 

<br>6. When not immediately used store the pellet at -20°C. 

<br>7. Resuspend the pellet in 15 ml/g wet pellet Lysis Buffer

<br>8. Freeze-thaw at least 3 times to lyse the cells. 

<br>9. Break DNA using a homogenizer. 

<br>10. Spin the cell lysate for 20 min at 12,000 rpm using a SS-34 rotor at 4 oC. Collect supernatant. Wash pellet with 10 ml lysis buffer per gram of pellet, homogenize and spin again. Collect and combine supernatant. 

<br>12. Spin supernatant at 25000 rpm for 15~25 min, 4 oC, using ultracentrifuge (be very careful). 

<br>13. Combine and apply the supernatant to 15~25 ml Ni column. Collect flow through. Apply flow through to the column again. 

<br>14. Wash column with 400 ml washing buffer 1. 

<br>15. Elute protein with 12 X 5 ml elution buffer 1 (Buffer B, with 1 mM b-ME, 300 mM imidazole). 

<br>16. Load eluate to 10-ml glutathione-agarose column.

<br>17. Wash with 150 ml PBS or 20mM Tris-HCl, with 150 mM NaCl, pH 7., until no protein is&nbsp; eluted (monitored using OD280).<br> 

<br>18. Elute GST-3C with 10 mM reduced glutathione in 20 mM TBS 20mM Tris-HCl, 150 mM NaCl, pH 7.5 (elution buffer 2) 

<br>19. Collect the eluate in 2 ml fractions. Measure protein conc in each fraction. Combine fractions containing GST-3C. Run SDS-PAGE to check purity, if not pure, purify further with TSK gel filtration column after concentrating using spin column (step 20).<br>

<br>20. Concentrate protein using Pierce 10kDa spin column, spin at 2500 rpm for 45 min, at 4 °C cold room. 

<br>21. Transfer concentrated protein solution to a dialysis tube, dialyze overnight against 40 ml storage buffer at 4°C. 

<br>22. Dialysis again with 40 ml Storage Buffer at 4°C. 

<br>23. Measure protein concentration, adjust DTT concentration to 1 mM final conc using 1 M or 2 M DTT. Transfer 1.5 ml to microcentrifuge tubes, print label (name, clone id, conc, lot number), store the aliquots at -80°C.<br>'''<br>''' 

'''Lysis Buffer'''<br> 

50 mM Tris-HCl pH 8.0 <br>300 mM NaCl <br>10% (v/v) glycerol <br>0.2% (v/v) NP-40<br> 

10 mM Imidazole<br>1 mM b-ME<br> 

<br> 

'''Wash Buffer '''<br> 

20 mM Tris-HCl pH 8.0<br>300 mM NaCl<br> 

10 mM Imidazole<br>5% (v/v) glycerol (optional)<br>1 mM b-ME<br> 

<br> 

'''Elution Buffer 1'''<br> 

20 mM Tris-HCl pH 8.0<br>300 mM NaCl<br>300 mM Imidazole<br> 

10% (v/v) glycerol<br>1 mM b-ME<br> 

<br> 

<br> 

'''Elution Buffer 2'''<br> 

50 mM Tris-HCl pH 8.0 <br>300 mM NaCl <br>10 mM reduced glutathione <br>10% (v/v) glycerol <br>1 mM b-ME

<br> 

'''Storage Buffer'''<br> 

50 mM Tris-HCl, pH 8.0<br>150 mM NaCl<br>10 mM EDTA<br>50% (v/v) glycerol<br>0.1 mM DTT 

<br><br> 

<br>