Editing His-Tat-NLS-Cre-Purification (section)

== '''Previous Working Protocol''' ==

'''Materials'''<br/>50% Ni-NTA agarose (Qiagen).<br/>Econo-Pac columns (Bio-Rad).<br/>Benzonase (Novagen).<br/>ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.<br/>Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.<br/>Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.<br/>Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).



'''Note:'''

'''Cre''': pI/Mw: 10.30 / 42764.07

*Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
*'''Step <u>7 to 24 </u>should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 11 and the yield will be very low if TT buffer/glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.



<br/>'''Buffers'''

<u>'''Buffer Q'''</u>: 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 1 mM EDTA.

'''Buffer Q2''': 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 1 mM EDTA.

<u>'''Suspension Buffer'''</u>: 50 mM Phosphate Buffer, pH 7.4,&nbsp;&nbsp; 500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

'''<u>Buffer A</u>''', 50 mM Phosphate Buffer, pH 7.4, 600 mM NaCl, '''20 mM '''imidazole, '''0.05 mM''' EDTA,&nbsp;&nbsp; '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' Store at 4 oC or in cold room.

'''4 L:'''lute Cre pro

190 ml of&nbsp;0.2 M NaH<sub>2</sub>PO<sub>4 </sub>(Dilute from 2 M NaH<sub>2</sub>PO<sub>4</sub>)

810 ml of 0.2 M Na<sub>2</sub>HPO<sub>4</sub>&nbsp; (dilute from 0.5 M Na<sub>2</sub>HPO<sub>4</sub>)

140 g NaCl

2.7 g Imidazole

2 ml 0.1 M EDTA

dd water to 4 L

Before use: add 70 ul beta-ME to 1 L

<br/>'''<u>TT buffer</u>''': 2M&nbsp; disodium tartrate. Store at 25 oC<br/>'''<u>Ni Washing buffer</u>:''' the same as Buffer A. Store at 4 oC or in cold room.<br/>'''<u>Ni Elution buffer</u>''':&nbsp; 50 mM Phosphate Buffer, pH 7.4, 150 mM NaCl, 20 mM imidazole, 0.05 mM EDTA,&nbsp; 250 mM imidazole, 250 mM Arginine, 10% glycerol. Store at 4 oC or in cold room.

'''<u>Cre Dilution Buffer:</u>''' 50 mM Phosphate Buffer,&nbsp; pH 7.4,&nbsp; 1 mM beta-ME, '''0.1 mM EDTA'''. Store at 4 oC or in cold room.

'''<u>SP Washing Buffer</u>'''<u>:</u> 50 mM Phosphate Buffer, pH 7.4, 500 mM NaCl,&nbsp; '''0.1 mM&nbsp;EDTA'''.&nbsp; Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.



'''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.1 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.



'''<u>SP Elution Buffer</u>''': 50 mM Phosphate Buffer, pH 7.4, 1 M NaCl,&nbsp; 250 mM Arginine,&nbsp; '''0.1 mM EDTA'''.&nbsp; Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''MEP Equilibration Buffer''': 20 mM Phosphate Buffer, pH 7.4, 1M NaCl, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Washing Buffer''': 20 mM Phosphate Buffer, pH 6.0,&nbsp; Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Elution Buffer''': 50 mM NaOAc, pH 5.0, '''300 mM''' Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

<br/>'''<u>Dialysis Buffer</u>''': 50% glycerol, 500 mM NaCl, 20 mM '''HEPES''' or 50 mM '''Na-Phophate '''Buffer,&nbsp; pH 7.4, 100 mM Arginine, '''0.1 mM EDTA'''. Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use Store at 4 oC or in cold room.

'''<u>1 M Arginine:</u>'''&nbsp; 174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

<br/>1. Transform&nbsp; Nico21(DE3)/plysN with the Cre expression plasmid 1708 pT7A3-HTNCre

2. Make 7 L '''LB'''

3. Inoculate 200 ml LB medium supplemented with 1% (w/v) glucose, 50 ug/ml '''Kan''', and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate <strike>TB or</strike> LB medium supplemented with 1% (w/v) '''glucose''',&nbsp; 50 ug/ml '''Kan''', and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of <strike>0.3 to 0.5</strike> 0.1 mM (add 0.1&nbsp; ml of 1 M IPTG to 1 L) and induce&nbsp; o/n at 25~ 30 oC.

6. Pellet cells by centrifugation (6000 rpm, 4 min) and freeze the bacterial pellets at −20 oC, or -80 oC, or dry ice. The cell pellets can be stored at −20 oC for several days, but it is advisable to use them as soon as possible.

7. Thaw pellets in warm water and resuspend in&nbsp; '''15 ml Buffer Q per liter culture '''using a magnetic plastic stick. Repeat freeze-thawing if cells are not lysed after one freezing-thawing.



8. Homogenize for 1&nbsp; min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more equal vol mix of '''Buffer Q'''&nbsp; if it is still sticky, optioally add 25 U/ml benzonase or DNase I to the suspension and incubate for 30 min at room temperature. Sonicate the suspension on ice for 2 min (power 50%, cycles 5) if it is still sticky.

<br/>9. Centrifuge the lysate for 20 min at 4 oC and&nbsp; 12000 rpm (do not run > 12000 rpm).

<u>Spin again at 25,000 rpm for 25 min at 4 oC on ultracentrifuge. Check if any one of the centrifuge tubes is broken if vaccum can not go down. If one of the tubes is broken, or seal is missing or cap is not tighten, vaccum will not go down and the centrifuge vaccum pump will be damaged by evaporated salt and water.</u>

Make sure ultracentrifuge tubes are not broken, tighten tubes before placing them into rotor. Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.



10. Decant the supernatant, load to a QFF (2.5×40 cm XK25) column couple to a SP FF (2.5×20 cm,&nbsp; XK25, 30~50 ml)&nbsp; column.

11. Wash QFF/SP FF columns with 500 to 1000 ml '''Buffer Q2 '''until OD280 drop to baseline.

12. Disconnect QFF. Wash SP&nbsp;FF column with '''SP Washing Buffer''' until OD280 drop to baseline.

13. Elute protein '''directly''' to 15~ 20 ml Ni column (EDTA compatible resin from Roche) using 150~ 200 ml of '''SP Elution Buffer '''at flow rate 1~3 ml/min.

Cre will be eluted at ~ 0.8 M NaCl at pH 7.5.

14. Wash column with 10 CV of Buffer A.

'''Optional: '''remove Endotoxin before elution:

Wash column with 100 ml of ETR Buffer to remove endotoxin

or

Wash the Ni beads 3 times with 2 CV of 0.1 M Arginine, or 2 times with 2 CV of 0.5 M Arginine.



After step 14,&nbsp; clean Q FF and SP FF columns with 2 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.





'''Optional steps 15 to 17'''

15. Elute Cre protein directly into a 15 ml MEP column by reverse-flow using&nbsp;'''<u>Ni Elution buffer.</u>'''

Ni column can be regenerated using EDTA, Urea, and then NiCl<sub>2</sub>.

Cre will bind to MEP at 1 M NaCl.



16. Wash MEP column with '''MEP Washing Buffer'''

17. Elute Cre protein by reverse-flow using '''MEP Elution Buffer'''. Collect 5 ml fraction. Combine fractions containing Cre protein.&nbsp; Measure protein using nanodrop.

Cre can be eluted from MEP column at pH 5.0, 300 mM Arginine.

MEP column can be regernated using 0.1 N Acetic acid, water, 0.1 N NaCl.



18. Elute protein using'''Ni Elution buffer.''' Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).&nbsp; Concentrate protein to ~ 5 to 10 mg using Pierce 9k spin columns.



19. Wash a dialysis tube thoroughly with ddH2O,&nbsp; Check if the dialysis tube is leaking.



20. Transfer Cre protein solution to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT, top cube of 4 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein in&nbsp; 70 ml of '''<u>Dialysis Buffer&nbsp;</u> '''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.



21. Wash dialysis tube with 2 X 0.5 ml of '''1 M''' arginine, combine proteins.



22. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.&nbsp;&nbsp; '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2&nbsp; mg/vial.''' &nbsp; The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.



23. Put vials immediately on ice.&nbsp; Make sure the protein is '''at least 1 mg per vial''' (give 1.2 mg so customers will not complain). Create a new lot number in the product database, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,&nbsp; number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.



24. Store the HTNCre vials in a freezer box at −20 oC o/n.



25. Place the freezer box in a foam box, put the foam box in −80 <sup>o</sup>C freezer. After 12 hours, take the foam box out and store the freezer box in -80 <sup>o</sup>C freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.



'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''