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

=== <br/>'''Buffers''' ===

<u>'''Buffer Q'''</u>: 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 50 mM NaCl, 1 mM EDTA.

<u>'''Buffer Q2'''</u>: 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 50 mM NaCl, 1 mM EDTA.

<u>'''Suspension Buffer'''</u>: 25 mM Tris-HCl, pH 8.0, &nbsp;&nbsp; 500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

<u>'''Lysis Buffer''':</u>&nbsp;25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol,&nbsp; 1 mM beta-ME.

'''<u>Buffer A</u>''', 25 mM Tris-HCl, pH 8.0,&nbsp; 500 mM NaCl,&nbsp;'''10 mM&nbsp;'''imidazole,&nbsp;'''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.'''&nbsp;check pH ~8. Store at 4 oC or in cold room.

<u>'''Buffer A2'''</u>, 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.



'''<u>TT buffer</u>''':&nbsp;

tartaric salt buffer:<br/>1.5 M L-Tartaric acid (Disodium salt)<br/>50 mM phosphate buffer, pH 7.4<br/>20 mM imidazole

Store at 25 oC

<br/>'''<u>Ni Washing buffer</u>:'''&nbsp;the same as Buffer A. Store at 4 oC or in cold room.<br/>'''<u>Ni Elution buffer</u>''':&nbsp; 50 mM Phosphate Buffer,&nbsp;'''pH 7.0''',&nbsp;'''250 mM NaCl''',&nbsp;'''100 mM arginine,'''&nbsp;&nbsp;&nbsp;'''300 mM imidazole''',&nbsp; 10% glycerol. Store at 4&nbsp;<sup>o</sup>C or in cold room.

'''<u>Cre Dilution Buffer:</u>'''&nbsp;50 mM Phosphate Buffer,&nbsp; pH 7.0,&nbsp; 1 mM beta-ME,&nbsp;'''0.1 mM EDTA'''. Store at 4 oC or in cold room.

<br/>'''ETR Buffer''':&nbsp;'''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 Dilution Buffer'''</u>''':&nbsp;'''50 mM Phosphate Buffer, pH 7.0,&nbsp; 0.1 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

'''<u>SP Washing Buffer</u>'''<u>:</u>&nbsp;50 mM Phosphate Buffer, pH&nbsp;'''7.0''',&nbsp;'''500 mM NaCl''',&nbsp;&nbsp;'''0.1 mM&nbsp;EDTA'''.&nbsp; Adjust to final concentration of&nbsp;'''1 mM'''&nbsp;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 Tris-HCl, pH&nbsp;'''8.0''',&nbsp;'''1.5 M'''&nbsp;NaCl,&nbsp; 5% glycerol,&nbsp;&nbsp;&nbsp;'''0.1 mM EDTA'''.&nbsp; Adjust to final concentration of&nbsp;'''1 mM'''&nbsp;from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use, check pH to see if the buffer pH is ~8.0. Store at 4 oC or in cold room.

'''<u>Dialysis Buffer</u>''': 50% glycerol, 500 mM NaCl, 20 mM&nbsp;'''HEPES'''&nbsp;or 50 mM&nbsp;'''Na-Phophate&nbsp;'''Buffer,&nbsp; pH 7.4, 100 mM Arginine,&nbsp;'''0.1 mM EDTA'''. Adjust to final concentration of&nbsp;'''1 mM'''&nbsp;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, for example, 1708 pT7A3-HTNCre

2. Make 8 L&nbsp;'''LB'''

3. Inoculate 200 ml LB medium supplemented with 0.5% (w/v) glucose, 50 ug/ml&nbsp;'''Kan''', and&nbsp;'''34 ug/ml chloramphenicol'''&nbsp;with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate&nbsp; LB medium supplemented with 0.5% (w/v)&nbsp;'''glucose''',&nbsp; 50 ug/ml&nbsp;'''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&nbsp; 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. Spin down cells,&nbsp; 5 min at 6000 rpm, RT.&nbsp; Add 100 ml Lysis Buffer to pellet and store at -80 oC.<br/>7. Thaw cell pellet, add ~400 ml&nbsp;'''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add&nbsp; freshly prepared lysozyme to final conc 1 mg/mL,&nbsp; and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).<br/>9.&nbsp; Incubate at room temperature for a few hours, or cold room overnight, until the cells are lysed.

10.&nbsp; Add equal volume of&nbsp;'''TT buffer''.'''''

'''''<font color="red">The folling steps (11 to 19) should be completed in 1 working day.</font>'''''

<br/>'''''11. Homogenize or sonicate on ice, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more buffer'''''<i>(equal vol mix of Buffer A and&nbsp;TT buffer)'</i>''',&nbsp;'''then homogenize on ice again.

If it is still too viscous (sticky)

Add more&nbsp; benzonase or DNase I&nbsp; to the lysate and incubate for 30 min at room temperature. Sonicate the suspension on ice for 1 min (power 50%, cycles 5)



'''Note:'''

It was reported that sonication for 1 min with probe will cause Cre to form soluble aggregate. It is better to avoid homegenization and sonication. Use water bath-type of&nbsp; mild sonicator is recommended after lysozyme and DNase 1 digestion:

30 s on/30 s off pulses, at low intensity for 15 minutes

Ref:&nbsp;[http://www.biomedcentral.com/1472-6750/9/80 http://www.biomedcentral.com/1472-6750/9/80]<br/><br/>12.&nbsp;&nbsp;<u>Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.</u>

<u>If vaccum can not go down:</u>

*<u>stop centrifuge.</u>
*<u>Check if centrifuge tube is broken or missing a seal ring.</u>
*<u>Check if centrifuge caps are tighten</u>

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

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.

13. Resuspend pellet with 200 ml of Lysis Buffer + TT Buffer (100 ml&nbsp;'''Lysis Buffer'''&nbsp;+ 100 ml&nbsp;'''TT buffer)'''. Homogenize and spin at 25000 rpm again.

14. Combine supernatant and&nbsp; load to a 10 ~15 ml Ni&nbsp; column.

15. Wash&nbsp; column with 200~ 500 ml&nbsp;'''Buffer A&nbsp;'''until OD280 drops to baseline.

16. Wash column with 1 CV of&nbsp;&nbsp;'''Buffer A2'''.

17. Elute protein with&nbsp; 150 ml or more of&nbsp;&nbsp;'''Ni Elution Buffer&nbsp;<font color="red">(not Buffer B)</font>&nbsp;directly to'''&nbsp; 10 ml&nbsp;'''Q FF&nbsp;'''column (Pharmacia) connected to a column packed with 30 ml&nbsp;'''SP FF''', preequilibrated with'''&nbsp;SP Dilution Buffer. Q FF is required for removing endotoxin.'''

18. Wash Q FF&nbsp;+ SP FF columns with&nbsp;'''SP Dilution Buffer'''&nbsp;until OD280 reaches baseline, optionally , use FPLC to monitor washing.

19.&nbsp;'''Disconnect Q FF,'''&nbsp;Wash SP&nbsp;FF with 2~10 CV of&nbsp;'''SP Washing Buffer'''&nbsp;until OD280 reaches baseline, optionally , use FPLC to monitor washing.

20. Elute with 5~10 CV of&nbsp;&nbsp;'''SP Elution Buffer,&nbsp;'''collect 10 ml fractions

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.



21. &nbsp; Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).&nbsp; Combine&nbsp; fractions containing low protein concentration and concentrate proteins to ~2&nbsp; to&nbsp; 5&nbsp; mg using Pierce 9k spin columns. Combine fractions containing high protein concentration and spin-column concentrated protein solution.



22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,&nbsp; Check if the dialysis tube is leaking.



23. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to&nbsp;'''2 mM DTT'''&nbsp;concentration from 2 M DTT (in a box labeled as AMP-DTT,&nbsp; -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with&nbsp; 45~50 ml of&nbsp;'''<u>Dialysis Buffer&nbsp;</u>&nbsp;'''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.



24. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

25. Change dialysis buffer and dialyse for another 8 hours or o/n

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 2 X 0.5 ml of&nbsp;'''1 M'''&nbsp;arginine, combine proteins.



27. 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;&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;&nbsp; The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50&nbsp;'''ug'''&nbsp;(not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use&nbsp;'''Dialysis Buffer II'''&nbsp;as control to test buffer toxicity.



28. Put vials immediately on ice. Create a new lot number in the protein and product databases, 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.



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



30. Place the freezer box in a foam box, put the foam box in −80&nbsp;<sup>o</sup>C freezer. After 12 hours, take the foam box out and store the freezer box in -80&nbsp;<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.'''



'''Materials'''<br/>50% Ni-NTA agarose (Qiagen).<br/>Econo-Pac columns (Bio-Rad).<br/>Benzonase (Novagen) without His tag or DNAse I.<br/>ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.<br/><s>Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.</s><br/><s>Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.</s><br/>Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).



'''Note:'''

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

*Add 0.001&nbsp;mM&nbsp; (1 uM) EDTA and 1 mM beta-ME to all buffers
*'''<font color="red">Step <u>11 to 19 </u>should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)</font>'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 25 and the yield will be very low if 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.
*<s>Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.</s>