His-Tat-NLS-Cre-Purification

Current Working Protocol

Lysis Buffer:  25 mM   KPO4 , pH 7.5, 500 mM NaCl, 0.1% NP-40, 10 mM imidazole, 10% glycerol,  1 mM beta-ME.

Buffer A,   25 mM KPO4 , pH 7.5,  500 mM NaCl, 10 mM imidazole, 10% glycerol.  Store at 4 oC  .

Ni Washing buffer: the same as Buffer A. Store at 4 oC .
Ni Elution buffer:  50 mM K Phosphate Buffer, pH 7.5, 200 mM NaCl,    150 mM imidazole,  10% glycerol. Store at 4 ^oC or in cold room.

Ni Elution buffer II:  50 mM K Phosphate Buffer, pH 7.5, 100 mM Arginine or Histidine,    150 mM imidazole,  10% glycerol. Store at 4 ^oC or in cold room.---Need to test

Cre Dilution Buffer: 50 mM K Phosphate Buffer,  pH 7.5,  100 mM arginine, 10% glycerol.  Store at 4 oC.

SP Washing Buffer: 50 mM K Phosphate Buffer,  pH 7.5,  10% glycerol.  Store at 4 oC .

SP Elution Buffer: 50 mM Phosphate Buffer, pH 7.5 , 1.5 M NaCl,  100 mM arginine, 10% glycerol.  check pH to see if the buffer pH is ~8.0. Store at 4 oC .

Dialysis Buffer: 50% glycerol, 500 mM NaCl, 20 mM HEPES,  pH 7.4, 100 mM Arginine, 0.1 mM EDTA.   1 mM  DTT, Store at 4 oC or in cold room.

1 M Arginine:  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.

1. Transform  BL21 star (DE3) with pET28b-Cre (Clone # 374), plate on LB/Kan, grow on 37 oC o/n.

2, Pick a few well isolated colonies, grow in 200 ml LB/Kan with shaking overnight at 37 oC.  Make Glycerol stock. 

3. Make 20 L  LB-G medium

310 g 2xYT  powder

150g NaCl

40 g Peptone or tryptone

200 ml glycerol.

autoclave, use 8 L  for each purification.

4. Aliquote  the overnight culture in a ratio of 1:50 to 1 L  LB-G medium and incubate with vigorous shaking at 37 oC until an OD600 > 1, usually 2~4 hours

5. Induce Cre expression by adding 1 M IPTG solution to a final concentration of   1  mM (add  1 ml of 1 M IPTG to 1 L) and induce   2~4 hours at 37 oC, or 0.2 mM IPTG o/n at 25~ 30 oC.

 6. Spin down cells,  6 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database. Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml cold Lysis Buffer.

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

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).

9.  Incubate for  30 ~90 min with stiring at room temperature, or cold room (4 oC) overnight, until the cells are lysed, and DNA is digested

10  Spin again at 25,000 rpm for 35 min at 4 oC on ultracentrifuge.

If vaccum can not go down, or speed can not go up to > 3000 rpm

• stop centrifuge.
• Check if centrifuge tube is broken or missing a seal ring.
• Check if centrifuge caps are tighten

Note:

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

Rotor should be  cooled down using dry ice. Remove residual water with clean paper or cloth .

11. Resuspend pellet with 200 ml of Lysis Buffer. homogenize for 10 sec and spin at 30000 rpm again.

12. Combine supernatant and  load to a 50 -100 ml Ni  column.

13. Wash  column with 200~ 1000 ml Lysis Buffer until OD280 drops to baseline.

Wash column with 2 bed vol of 1.5 M NaCl, 50 mM K-PO4, pH 7.5 

14. Wash column with 1 CV of  Buffer A.

15. Elute protein with  200 ml or more of  Ni Elution Buffer. (350 ml)

16. Dilute eluted protein solution with 3 volume of  Cre Dilution Buffer (1000 ml). Watch solution carefully, if protein is aggregating (protein solution turns to turbid), add solid arginine and stire immediately until the solution turns to clear! 

17. Load to a 10 ml Q FF column (Pharmacia) connected to a column packed with 30~50 ml Capto  SP ImpRes or  SP FF or SP HP or 20 ml , preequilibrated with Cre Dilution Buffer. Q FF is required for removing endotoxin.

Dilute eluant with > 2 volume of SP Dilution Buffer, load to Q FF + SP FF columns using P1 pump + air bubble detector.

18. Wash Q FF + SP FF columns with Cre Dilution Buffer or SP Washing Buffer until OD280 reaches baseline, optionally , use FPLC to monitor washing.

optional: Disconnect Q FF.

19. Elute with 5~10 CV of  SP Elution Buffer  by reverse flow (invert column), collect 5 ml fractions, 

optionally, elute using linear gradient from  Cre Dilution Buffer to SP Elution Buffer, 10 CV

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

20.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml). 

Combine fractions containing high protein concentration

21. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

22. 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 2 mM DTT concentration from 2 M DTT (in a box labeled as AMP-DTT,  -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  45~50 ml of Dialysis Buffer  (in 100 ml beaker) in fridge, mix occasionally for several times, then dialyse overnight to concentrate protein, Measure OD280. 

For freeze drying: use Dialysis Buffer A, otherwise use Dialysis B

23. 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 (for longterm storage, wash with 20% ethanol).

24. Next morning, change Dialysis Buffer, dialyse for additional 1~6 hours, mix occasionally 

25. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3 X 1 ml of 1 M arginine, combine proteins.

26. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at 1~1.2  mg/vial.   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 as control to test buffer toxicity.

27. 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,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

38. Place the freezer box in a freezer box,    store the freezer box in -80 ^oC freezer.  

39. Wash Dialysis tube with water, store in  20% ethanol in a bottle container

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

Current Working Protocol for  Product in Liquid Form

Materials
50% Ni-NTA agarose (Qiagen).
Econo-Pac columns (Bio-Rad).
Benzonase (Novagen).
ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.
Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.
Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.
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 11 to 19 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 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.
• Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

Buffers

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

Buffer Q2: 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 50 mM NaCl, 1 mM EDTA.

Suspension Buffer: 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol,  1 mM beta-ME.

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

Buffer A2, 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.

TT buffer: 

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

Store at 25 oC

Ni Washing buffer: the same as Buffer A. Store at 4 oC or in cold room.
Ni Elution buffer:  50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine,   300 mM imidazole,  10% glycerol. Store at 4 ^oC or in cold room.

Cre Dilution Buffer: 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, 0.1 mM EDTA. 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.

SP Dilution Buffer: 50 mM Phosphate Buffer, pH 7.0,  0.1 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

SP Washing Buffer: 50 mM Phosphate Buffer, 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.

SP Elution Buffer: 50 mM Tris-HCl, pH 8.0, 1.5 M NaCl,  5% glycerol,   0.1 mM EDTA.  Adjust to final concentration of 1 mM 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.

Dialysis Buffer: 50% glycerol, 500 mM NaCl, 20 mM HEPES or 50 mM Na-Phophate Buffer,  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.

1 M Arginine:  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

1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre

2. Make 8 L LB

3. Inoculate 200 ml LB medium supplemented with 0.5% (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  LB medium supplemented with 0.5% (w/v) glucose,  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  0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Spin down cells,  5 min at 6000 rpm, RT.  Add 100 ml Lysis Buffer to pellet and store at -80 oC.
7. Thaw cell pellet, add ~400 ml cold Lysis Buffer.

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

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

10.  Add equal volume of TT buffer.

The folling steps (11 to 19) should be completed in 1 working day.

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

If it is still too viscous (sticky)

Add more  benzonase or DNase I  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  mild sonicator is recommended after lysozyme and DNase 1 digestion:

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

Ref: http://www.biomedcentral.com/1472-6750/9/80

12.  Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down:

• stop centrifuge.
• Check if centrifuge tube is broken or missing a seal ring.
• Check if centrifuge caps are tighten

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 Lysis Buffer + 100 ml TT buffer). Homogenize and spin at 25000 rpm again.

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

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

16. Wash column with 1 CV of  Buffer A2.

17. Elute protein with  150 ml or more of  Ni Elution Buffer (not Buffer B) directly to  10 ml Q FF column (Pharmacia) connected to a column packed with 30 ml SP FF, preequilibrated with SP Dilution Buffer. Q FF is required for removing endotoxin.

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

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

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

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

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  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,  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 2 mM DTT concentration from 2 M DTT (in a box labeled as AMP-DTT,  -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  45~50 ml of Dialysis Buffer  (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 1 M 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.   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  mg/vial.   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.

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,  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 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC 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
50% Ni-NTA agarose (Qiagen).
Econo-Pac columns (Bio-Rad).
Benzonase (Novagen) without His tag or DNAse I.
ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.
Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.
Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.
Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

Note:

Cre: pI/Mw: 10.30 / 42764.07

• Add 0.001 mM  (1 uM) EDTA and 1 mM beta-ME to all buffers
• Step 11 to 19 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 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.
• Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

Buffers

Buffer Q: 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 10% glycerol, 50 mM NaCl, 0.001 mM EDTA.

Buffer Q2: 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 50 mM NaCl, 0.001 mM EDTA.

Suspension Buffer: 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.001 mM EDTA, 1 mM beta-ME.

Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 5% glycerol,  1 mM beta-ME.

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

Buffer A2, 25 mM Tris-HCl, pH 8.0, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.

TT buffer: 

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

Store at 25 oC

Ni Washing buffer: the same as Buffer A. Store at 4 oC or in cold room.
Ni Elution buffer:  50 mM Phosphate Buffer, pH 7.0, 100 mM NaCl, 100 mM arginine,   150 mM imidazole,  10% glycerol. Store at 4 ^oC or in cold room.

Cre Dilution Buffer: 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, 0.001 mM EDTA. Store at 4 oC or in cold room.

ETR Buffer: 200 mM Tris-HCl, pH 7.0, 500 mM NaCl, 0.001 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.

SP Dilution Buffer: 50 mM Phosphate Buffer, pH 7.0,  0.001 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

SP Washing Buffer (No glycerol): 50 mM Phosphate Buffer, pH 7.0, 200 mM NaCl,  0.001 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.

SP Elution Buffer (No glycerol): 50 mM Tris-HCl, pH 8.0, 1.5 M NaCl,  100 mM arginine, 15% Trehalose,  0.001 mM EDTA.  Adjust to final concentration of 1 mM 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.

Dialysis Buffer A (No glycerol): 500 mM NaCl, 10 mM HEPES,  pH 7.4, 100 mM Arginine,  15% Trehalose, 0.001 mM EDTA, add 1 mM DTT just before use. Store at 4 oC or in cold room.

Dialysis Buffer B (with glycerol): 500 mM NaCl, 10 mM HEPES,  pH 7.4, 100 mM Arginine,  50% glycerol, 0.001 mM EDTA, add 1 mM DTT just before use. Store at 4 oC or in cold room.

1 M Arginine:  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

1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre or 1010 pBad-Cre

2. Make 20 L  LB Plus medium

500 g LB powder

200 g yeast extract

80 g Na2HPO4.7H2O

10 g KH2PO4

40 g glucose

autoclave, use 8 L  for each purification.

3. Inoculate 200 ml   LB Plus  medium supplemented with   50 ug/ml Kan (for 1708) or 100 ug/ml Amp (for 1010) and 34 ug/ml chloramphenicol with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate  LB Plus medium supplemented with    50 ug/ml Kan  (for 1708) or 100 ug/ml Amp (for 1010)  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 > 0.4

5. Induce Cre expression by adding 1 M IPTG solution to a final concentration of  0.2 mM (add 0.2  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.---> for clone 1708

or  Induce expression by adding 1~2 g arabinose to 1 L   and induce  o/n at 30 ^oC,  or 6~8 hours at 37 ^oC---> for clone 1010

6. Spin down cells,  5 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database. Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml cold Lysis Buffer.

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

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).

9.  Incubate at room temperature for a few hours, or cold room overnight, until the cells are lysed, and DNA is digested

10.  Add equal volume of TT buffer (optional)

The folling steps (11 to 19) should be completed in 1 working day.

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

If it is still too viscous (sticky)

Add more  benzonase (no his tag) or DNase I  to the lysate and incubate for 30 ~90 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  mild sonicator is recommended after lysozyme and DNase 1 digestion:

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

Ref: http://www.biomedcentral.com/1472-6750/9/80

12.  Spin again at 30,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down, or speed can not go up to > 3000 rpm

• stop centrifuge.
• Check if centrifuge tube is broken or missing a seal ring.
• Check if centrifuge caps are tighten

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 Lysis Buffer + 100 ml TT buffer). Homogenize and spin at 25000 rpm again.

13. Resuspend pellet with 200 ml of Lysis Buffer . Homogenize for 10 sec and spin at 30000 rpm again.

14. Combine supernatant and  load to a 50 ml Ni  column.

15. Wash  column with 200~ 1000 ml Buffer A until OD280 drops to baseline.

16. Wash column with 1 CV of  Buffer A2.

17. Elute protein with  150 ml or more of  Ni Elution Buffer (not Buffer B) directly to  10 ml Q FF column (Pharmacia) connected to a column packed with 30~50 ml SP FF, preequilibrated with SP Dilution Buffer. Q FF is required for removing endotoxin.

Optionally, elute Cre into 10 ml fractions with Ni Elution Buffer, wach the color of eluant while the protein is eluting out from column,  if you see protein is aggregating (solution is becoming turbid), add  a few ml of 1 M arginine and shake gently until solution is clear.  Dilute eluant with 2 volume of SP Dilution Buffer, load to Q FF + SP FF columns using P1 pump + air bubble detector.

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

19. Wash Q FF+ SP FF with 2 CV of SP Washing Buffer until OD280 reaches baseline,   use FPLC to monitor washing. 

optional: Disconnect Q FF.

20. Elute with 5~10 CV of  SP Elution Buffer 'by reverse flow', collect 10 ml fractions, 

optionally, elute using linear gradient from  SP Washing Buffer to SP Elution Buffer, 10 CV

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

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml). 

optionally, combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  mg using Pierce 9k spin columns.

Combine fractions containing high protein concentration and spin-column concentrated protein solution.

Optional: Dilute combined fractions with 10 volumes of SP Dilution Buffer. Load to 10~20 ml heparin sepharose column. Wash extensively with SP Dilution Buffer. Then elute Cre using linear gradient from SP Dilution Buffer to SP Elution Buffer<u</u>

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  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 2 mM DTT concentration from 2 M DTT (in a box labeled as AMP-DTT,  -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  45~50 ml of Dialysis Buffer  (in 100 ml beaker) in fridge, mix occasionally for several times, then dialyse overnight to concentrate protein, Measure OD280. 

For freeze drying: use Dialysis Buffer A, otherwise use Dialysis B

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 (for longterm storage, wash with 20% ethanol).

25. Next morning, change Dialysis Buffer, dialyse for additional 3~6 hours, mix occasionally 

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3 X 1 ml of 1 M 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.   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  mg/vial.   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 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,  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 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC 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.

Current Working Protocol for  Product in Liquid Form

Materials
50% Ni-NTA agarose (Qiagen).
Econo-Pac columns (Bio-Rad).
Benzonase (Novagen).
ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.
Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.
Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.
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 11 to 19 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 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.
• Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

Buffers

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

Buffer Q2: 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 50 mM NaCl, 1 mM EDTA.

Suspension Buffer: 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol,  1 mM beta-ME.

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

Buffer A2, 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.

TT buffer: 

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

Store at 25 oC

Ni Washing buffer: the same as Buffer A. Store at 4 oC or in cold room.
Ni Elution buffer:  50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine,   300 mM imidazole,  10% glycerol. Store at 4 ^oC or in cold room.

Cre Dilution Buffer: 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, 0.1 mM EDTA. 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.

SP Dilution Buffer: 50 mM Phosphate Buffer, pH 7.0,  0.1 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

SP Washing Buffer: 50 mM Phosphate Buffer, 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.

SP Elution Buffer: 50 mM Tris-HCl, pH 8.0, 1.5 M NaCl,  5% glycerol,   0.1 mM EDTA.  Adjust to final concentration of 1 mM 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.

Dialysis Buffer: 50% glycerol, 500 mM NaCl, 20 mM HEPES or 50 mM Na-Phophate Buffer,  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.

1 M Arginine:  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

1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre

2. Make 8 L LB

3. Inoculate 200 ml LB medium supplemented with 0.5% (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  LB medium supplemented with 0.5% (w/v) glucose,  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  0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Spin down cells,  5 min at 6000 rpm, RT.  Add 100 ml Lysis Buffer to pellet and store at -80 oC.
7. Thaw cell pellet, add ~400 ml cold Lysis Buffer.

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

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

10.  Add equal volume of TT buffer.

The folling steps (11 to 19) should be completed in 1 working day.

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

If it is still too viscous (sticky)

Add more  benzonase or DNase I  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  mild sonicator is recommended after lysozyme and DNase 1 digestion:

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

Ref: http://www.biomedcentral.com/1472-6750/9/80

12.  Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down:

• stop centrifuge.
• Check if centrifuge tube is broken or missing a seal ring.
• Check if centrifuge caps are tighten

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 Lysis Buffer + 100 ml TT buffer). Homogenize and spin at 25000 rpm again.

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

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

16. Wash column with 1 CV of  Buffer A2.

17. Elute protein with  150 ml or more of  Ni Elution Buffer (not Buffer B) directly to  10 ml Q FF column (Pharmacia) connected to a column packed with 30 ml SP FF, preequilibrated with SP Dilution Buffer. Q FF is required for removing endotoxin.

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

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

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

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

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  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,  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 2 mM DTT concentration from 2 M DTT (in a box labeled as AMP-DTT,  -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  45~50 ml of Dialysis Buffer  (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 1 M 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.   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  mg/vial.   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.

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,  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 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC 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.

Purification of Cre using HiTrap Heparin 

To prepare 1 M K-Phosphate: 300 ml of 1 M KH2PO4, 700 ml 1M K2HPO4, the pH is 7.0 measured using a pH meter.

Buffer A: To make 20 mM K-Phophate, 5% glycerol: 40 ml 1 M K-Phosphate Buffer, pH 7.0, 100 ml glycerol, add ddH2O to 2 L.

Buffer B: To make  20 mM K-Phophate, 1.5 M NaCl, 5% glycerol: dissolve 87.66 g NaCl in H2O,  40 ml 1 M K-Phosphate Buffer, pH 7.0, 100 ml glycerol, add ddH2O to 2 L

Column: 1 ml column, equilibrate with 20 mM Potassium Phosphate Buffer, pH 7.0.

Load 150 ml sample containing ~100 mM KCl (1:10 dilution from  sample eluted from SP FF using 1 M KCl, pH 7.0).

Linear gradient 15 ml from Buffer A to B, 1.5 ml/min, 3 ml/fraction. OD260 nanodrop: 0.8 mg/ml, total 2.4 mg in fraction 6.

Look like Cre is eluted at 0.7 M NaCl, when pH is 7.0

Date:5/19/2019, by Paul Xu

Purification of Cre using SOURCE 30S

30S-D

2.3 g dibasic NaPO4, 2.6 g monobasic KPO4
or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4)
15 g NaCl, 0.25 M
50g glycerol
(check pH to see if it is ~ 6.8)
Fill to 1 L with diH2O

30S-W

2.3 g dibasic NaPO4, 2.6 g monobasic KPO4
or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4)
30 g NaCl, 0.5 M
50g glycerol
(check pH to see if it is ~ 6.8)
Fill to 1 L with diH2O

30S-E
1.2 g dibasic NaPO + 1.3 g monobasic KPO4
or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4)
88 g NaCl, 1.5 M
25 g glycerol
(check pH to see if it is ~ 6.8)
Fill with diH2O to 1 L

1. Mix protein solution eluted from Ni column with equal volume of Buffer 30S-D.

2. Load protein to 15 ml SOURCE 30S column, pre-equilibrated with Buffer 30S-W.

3. Wash column with 30S-W until no protein is detectable (OD280 <0.05).

4. Elute Tat-Cre with Buffer 30S-E

Previous Working Protocol

Materials
50% Ni-NTA agarose (Qiagen).
Econo-Pac columns (Bio-Rad).
Benzonase (Novagen).
ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC.
Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.
Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.
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 7 to 24 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.

Buffers

Buffer Q: 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.

Suspension Buffer: 50 mM Phosphate Buffer, pH 7.4,   500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

Buffer A, 50 mM Phosphate Buffer, pH 7.4, 600 mM NaCl, 20 mM imidazole, 0.05 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.

4 L:lute Cre pro

190 ml of 0.2 M NaH₂PO₄ (Dilute from 2 M NaH₂PO₄)

810 ml of 0.2 M Na₂HPO₄  (dilute from 0.5 M Na₂HPO₄)

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

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

Cre Dilution Buffer: 50 mM Phosphate Buffer,  pH 7.4,  1 mM beta-ME, 0.1 mM EDTA. Store at 4 oC or in cold room.

SP Washing Buffer: 50 mM Phosphate Buffer, pH 7.4, 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.

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.

SP Elution Buffer: 50 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  250 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.

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,  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.

Dialysis Buffer: 50% glycerol, 500 mM NaCl, 20 mM HEPES or 50 mM Na-Phophate Buffer,  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.

1 M Arginine:  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

1. Transform  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 TB or LB medium supplemented with 1% (w/v) glucose,  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 0.3 to 0.5 0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  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  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  min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more equal vol mix of Buffer Q  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.

9. Centrifuge the lysate for 20 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

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.

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,  XK25, 30~50 ml)  column.

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

12. Disconnect QFF. Wash SP 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,  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 Ni Elution buffer.

Ni column can be regenerated using EDTA, Urea, and then NiCl₂.

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

19. Wash a dialysis tube thoroughly with ddH2O,  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  70 ml of Dialysis Buffer  (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.   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  mg/vial.   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.  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,  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 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC 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.

Protocol 1

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

Note:

• Use Phosphate Buffer for purification. Do not use buffer closes to PI (pH>8).
• Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
• Step 7 to 24 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.

Buffers

Suspension Buffer: 50 mM Phosphate Buffer, pH 7.4,   500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

Buffer Q: 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 1 mM EDTA.

Buffer A, 50 mM Phosphate Buffer, pH 7.4, 600 mM NaCl, 20 mM imidazole, 0.05 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.

4 L:lute Cre pro

190 ml of 0.2 M NaH₂PO₄ (Dilute from 2 M NaH₂PO₄)

810 ml of 0.2 M Na₂HPO₄  (dilute from 0.5 M Na₂HPO₄)

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

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

Cre Dilution Buffer: 50 mM Phosphate Buffer,  pH 7.4,  1 mM beta-ME, 0.1 mM EDTA. Store at 4 oC or in cold room.

SP Washing Buffer: 50 mM Phosphate Buffer, pH 7.4, 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.

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.

SP Elution Buffer: 50 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  250 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.

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,  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.

Dialysis Buffer: 50% glycerol, 500 mM NaCl, 20 mM HEPES or 50 mM Na-Phophate Buffer,  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.

1 M Arginine:  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

1. Transform  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 TB or LB medium supplemented with 1% (w/v) glucose,  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 0.3 to 0.5 0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  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  15 ml Suspension Buffer per liter culture using a magnetic plastic stick. Repeat freeze-thawing if cells are not lysed after one freezing-thawing. Add equal volume of  cold TT Buffer.

8. Homogenize for 1  min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more equal vol mix of Suspension Buffer and TT buffer 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.

9. Centrifuge the lysate for 20 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

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.

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  30~50 ml SP FF column.

11. Wash SP FF column with 200~300 ml SP Washing Buffer

12. 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.

13. Wash column with 10 CV of Buffer A.

14. Wash column with 100 ml of ETR Buffer to remove endotoxin.

Optional: remove Endotoxin before elution:

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,  clean SP column 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 Ni Elution buffer.

Ni column can be regenerated using EDTA, Urea, and then NiCl₂.

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

19. Wash a dialysis tube thoroughly with ddH2O  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  70 ml of Dialysis Buffer  (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.   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  mg/vial.   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.  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,  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 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC 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.

Activity Assay

1. Transfect 203T cells in 24 well plate with reporer plasmid (0.5~1 ug)

 1522 CMV-Brainbow-2.1-R Plasmid (from Green to Red)

 2181: pCMV-LoxP-kozak-TurboRFP-ODC-2Stops-LoxP-TurboGFP (from Red to Green)

2. 12 hours after transfection, add Cre recombinase (sterize).

3. Take pictures under fluroencese microscope for 3~5 days, take a note of cell viability.

DO NOT USE

1. Convert protein concentration in mg/ml to molar concentration

Use this link: http://www.my-whiteboard.com/pub/html/mw_2_moles.html

2. Add Cre to LoxP reporter cell line (either stable cell line or 293T or 293E cells transfected with LoxP-GFP-LoxP-RFP) grown in  24 well plate, concentration 1, 2.5, 5, 7.5 and 10 uM.

3. Observe recombination efficiency using flourescent microscope.

Protocol 2

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

Note:

• Use Phosphate Buffer for purification. Do not use extreme buffer (pH>8).
• Step 7 to 17 should be completed within reasonable short time (Must be completed within a 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 (<100 mM NaCl) for a long time. Add arginine up to 0.5 M if low salt buffer is needed for purification.

Buffers
Buffer A, 50 mM Phosphate Buffer, pH 7.4, 500 mM NaCl, 10 mM imidazole,10% glycerol (adjust to 10% glycerol using used Dialysis Buffer II), 50 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.
TT buffer: 500 mM disodium tartrate.
Ni Washing buffer: the same as Buffer A.
Ni Elution buffer:  Buffer A with 250 mM imidazole. Store at 4 oC.

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, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

MEP Elution Buffer: 20 mM Phosphate Buffer, pH 6.0, 20 mM NaOAc, pH5.0, 50 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

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

SP Washing Buffer: 20 mM Phosphate Buffer, pH 7.4, 500 mM NaCl, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

SP Washing Buffer II: 200 mM Tris-HCl, pH 7.0~pH 7.4, 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.

SP Elution Buffer: 20 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  0.1 mM EDTA, 250 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

Dialysis Buffer I: 500 mM NaCl, 20 mM  Phosphate Buffer, pH 7.4, 50 mM Arginine.
Dialysis Buffer II: 50% glycerol, 500 mM NaCl, 20 mM Phosphate Buffer,  pH 7.4, 50 mM Arginine.

1 M Arginine:  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Arginine is very expensive.

Expression and Purification

1. Transform TUNER (DE3)pLacI (Novagen) Nico21 (DE3) with the Cre expression plasmid pTriEx-HTNC, pT7A3-HTNCre

pET28bCre: C43(DE3) pLysS or Nico(DE3)/pLysN

2. Make 7 L TB medium (1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol), or LB

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

pET28bCre: Kan/Chl for Nico(DE3)/pLysN

4. Inoculate LB medium supplemented with 1% (w/v) glucose, 100 ug/ml ampicillin, 50 ug/ml Kanamycin 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.

pET28bCre: Kan/Chl, 1% (w/v) glucose

5. Induce expression by adding 1 M IPTG solution to a final concentration of 0.1 mM IPTG 0.5 mM (add 0.5 ml of 1 M IPTG to 1 L) and continue incubation for 1 to 2 hours

2 hours. or 30 oC for 6 hours. 25 oC o/n. OD600 should be 2.3 after 2 hours at 37 oC for pET28bCre in C43(DE3) pLysS  cells

For Nico(DE3)/pLysN: induce with 0.1 mM IPTG o/n at 25 oC, or 6 hour at 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  20 to 30 ml 1X Buffer A per liter culture using a magnetic plastic stick.

pTriEx-HTNC/TUNER (DE3)pLacI: resuspend cells in 10 ml of 1X Buffer A (without 500 mM NaCl) per liter culture and then add lysozyme stock solution (80 mg/ml) to a final concentration of 2 mg/ml and incubate at room temperature with continuous stirring for 20 min.

9. Add equal volume of ice-cold TT buffer to lysed bacteria, mix well.

10. Homogenize for 1 to 2 min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more lysis buffer and TT buffer 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).

11. Centrifuge the lysate for 30 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

Spin again at 30000 rpm for 25 min at 4 oC on ultracentrifuge. Turn off centrifuge for 1 hour if vaccum can not go down, then turn on again.

12. Decant the supernatant, load to 2.5 cm column packed with 5 ~ 10 ml Ni beads.

Alternatively, the following steps can be used

• add 2 ml 50% Ni-NTA agarose per liter of expression culture (total 14 ml beads, or 28 ml 50% beads), mix well, and pour suspension into 50-ml tubes.

• Incubate the suspension with continuous rotation at 4 oC for 1 h.
• Spin down beads (1 min at 3000 rpm, 4 oC).
• Resuspend beads in lysis buffer and pour the suspension into Econo-Pac® columns (Bio-Rad) or Sigma glass columns and allow the liquid to flow through completely. 

13. Wash the Ni beads twice with 5 to 20 column volumes of NI Washing Buffer (10 mM imidazole) until no protein is detectable.

14. Equilibrate 5 (or 10) ml MEP column with MEP Equilibration Buffer.

15. Elution of the protein directly to the MEP column with 10 ~15 CV  MEP Elution Buffer,

16. Wash column with at least 10 to 15  CV of MEP Washing Buffer  until no protein is detectable.

17. Elute Cre protein directly to 5 (or 10  ml) of SP or CM column with 10 to 15 CV of MEP Elution Buffer. Close the column valve.

Note: add 1 vol of dilution buffer (20 mM Phosphate Buffer, pH 7.5) to eluate if elution to CM column. For SP column dilution is not needed.

After elution, turn on the column valve and adjust flow rate to ~ 1 ml/min so that the Cre protein can bind to SP or CM resin.

20. Wash SP or CM column with 4 CV of  CM Washing Buffer or SP Washing Buffer.

21. Elute Cre from SP or CM column with 5 to 10 CV of  CM Elution Buffer orSP Elution Buffer, collect 3 ml fractions. Add elution buffer and let the resin to soak in the elution buffer for 1 min before openning the valve. usually 10 fractions are enough.

22. Measure protein concentration in each fraction using a spectrophotomer. Combine fractions containing Cre.

23. After step 25 clean MEP column with 50 mM NaAc, pH 3.5, then wash beads with 10 CV of water then soak resin in 20% Ethanol (for long term storage) or TBS (pH 8.0) with 150 mM NaCl. MEP resin should be cleaned with 0.1 N NaOH after 3 ~ 5 uses.

24. After step 25 clean CM or SP column with 2 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

25. Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).

26. Wash a dialysis tube thoroughly with double-distilled water.  Check if the dialysis tube is leaking.

27. Transfer Cre protein solution to the dialysis tube. Adjust to 1 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. Dialyse protein in 70 ml of Dialysis Buffer I  (in 100 ml beaker) for 3~4 hour in cold room.  Measure OD280. Dialyze Cre protein again for 3~4 h in cold room. Measure OD280.

28. Dialyse with 50 ml of Dialysis Buffer II in cold room for 4 to 8 hours, mix protein sol with clean pippette every 2 ~ 4 hour. Change buffer then dialyse overnight to concentrate protein, Measure OD280.

29. Wash dialysis tube with 2 X 0.5 ml of 100 mM arginine, combine proteins.

30. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   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 mg/vial.   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.

31. Put vials immediately on ice.  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, number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

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

34. Place the freezer box in a foam box, put the foam box in −80 ^oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 ^oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

Cre protein sequence:

MGHHHHHHGM GAAGRKKRRQ RRRPPAGTSV SLKKKRKVSN LLTVHQNLPA LPVDATSDEV
RKNLMDMFRD RQAFSEHTWK MLLSVCRSWA AWCKLNNRKW FPAEPEDVRD YLLYLQARGL
AVKTIQQHLG QLNMLHRRSG LPRPSDSNAV SLVMRRIRKE NVDAGERAKQ ALAFERTDFD
QVRSLMENSD RCQDIRNLAF LGIAYNTLLR IAEIARIRVK DISRTDGGRM LIHIGRTKTL
VSTAGVEKAL SLGVTKLVER WISVSGVADD PNNYLFCRVR KNGVAAPSAT SQLSTRALEG
IFEATHRLIY GAKDDSGQRY LAWSGHSARV GAARDMARAG VSIPEIMQAG GWTNVNIVMN
YIRNLDSETG AMVRLLEDGD

Molecular Weight: 42763 (42. kDa), Extinction Coefficient: 49270. PI=10.30

OD280 X 0.87=mg/ml

Use this link to calculate protein concentration based on OD280:

http://www.excellgen.net/eln/tools/protein_tools.html

Use this link to calculate molecular weight and PI

http://web.expasy.org/cgi-bin/compute_pi/pi_tool

Cre Recombinase Transduction into ES Cells

1. Plate single-cell suspension of mES cells in normal ES medium with 10–15% fetal calf serum.

2. Prepare ES medium containing 10 uM HTNCre protein by diluting an appropriate amount of HTNCre glycerol stock solution with normal ES medium. Sterilize medium with 0.22-um filter.

3. After 5–6 h or when the cells are attached, wash carefully with PBS.

4. Incubate cells for 8–20 h with HTNCre-containing medium.

For serum-free ES medium, a 5- to 10-fold lower concentration of HTNCre is recommended because FCS inhibits the transduction

5. After protein transduction, wash once with PBS and add normal ES medium.

Cre Recombinase Transduction into Undifferentiated hES Cells

Culture hES cells in KO/SR medium on tissue culture plates coated with mitotically inactivated mouse embryonic fibroblasts (MEFs). Cells are passaged by manual scraping or collagenase IV treatment.

Note: Cre protein transduction into hES cells is most efficient if the colonies are very small, ideally clumps of few cells. Therefore, the hES cell colonies are dissociated using accutase II (PAA Laboratories) prior transduction. Accutase II has the advantage that it selectively detaches the hES cells from feeder cells if incubation does not exceed 30 min. The single-cell suspension is plated on MEFs again and allowed to adhere for 24 h. Within this time, very small hES cell colonies are formed, which have the optimal size for protein transduction.

Transduction efficiency is optimal for Cre concentrations of 6 uM and transduction times of 6 h. Longer incubation times do not significantly increase transduction efficiency. The optimal cell density for Cre protein transduction is about 50,000 hES cells per cm2 .

1. One day prior to protein transduction plate mitotically inactivated MEF cells on the desired number of tissue culture plates.

2. Remove the medium from the hES cells and add enough prewarmed accutase II solution to cover the colonies evenly. Incubate for 25 min at 37 oC.

3. Detach the cells with KO/SR medium and transfer them to a 50-ml centrifugation tube. Take a sample of 15 l and mix it with the equal volume of trypan blue solution to stain the cells. Take 15 l stained cells, count the cell number in a Fuchs-Rosenthal chamber, and calculate the total number of hES cells. Calculate how many cells are required for the desired number of tissue culture plates.

4. Transfer the calculated volume of the cell suspension to 15- or 50-ml centrifugation tubes and centrifuge for 5 min at 1000 × g and 4 oC.

5. Resuspend the pellet in KO/SR medium and dispense the cell suspension on the MEF cell-coated tissue culture plates. Allow the cells to adhere for 24 h at 37 oC and 5% CO2.

6. Dilute the Cre protein stock solution in KO/SR medium to the desired concentration.

7. Remove the KO/SR medium from the plated hES cells and add enough Cre-containing KO/SR medium to cover the cells evenly.

8. Incubate for 1–6 h at 37 oC and 5% CO2 .

9. Remove the Cre-containing medium, wash the cells once with knockout DMEM and add normal KO/SR medium again.

10. Prior further use of the Cre-transduced cells, allow the cells to recover for at least 24 h.

Cre Recombinase Transduction into hES Cell-Derived Neural Precursors

Prepare neural precursor cells from hES cells according to a protocol published by Zhang et al. (In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 2001 19, 1129–1133).

Transfer embryoid bodies (EBs) to PO-coated cell culture plates.

Propagate cells for 10 days in ITSFn medium. Within this time period, neural tube-like structures develop in the EB. These structures are isolated mechanically and are propagated as free-floating neurospheres in N2 medium for 2 weeks. Finally, isolate neural precursor cells by enzymatic dissociation of these neurospheres, plate on PO/laminin-coated culture plates and further cultivate in the presence of FGF2 according to Zhang's protocol. Alternatively, human neural precursors may be derived from fetal Central Nervous System (CNS) tissue based on Brüstle's protocol (1998, Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat. Biotechnol. 16,1040–1044). Optimal parameters for Cre transduction in ES cell-derived neural precursors are a Cre concentration of 1 uM and a transduction time of 6 h. For maximum survival, it is crucial that the cells are confluent before transduction.

1. Dilute the Cre protein stock solution in the medium used for the cultivation of neural precursors to a final concentration of 1 uM.

2. Remove the culture medium from the neural precursor cells and add the Cre protein solution. Incubate for 6 h at 37 oC and 5% CO2.

3. Wash the cells twice with DMEM : F12 medium, then add again the suitable culture medium.

4. Before further use, allow the cells to recover for at least 24 h.

Purification of Cre using MEP  column

1. After step 17, load supernatant to XK 16/20 Column packed with 10 ml Ni resin  using a peristaltic pump.

2. Wash column using 200 ml washing buffer containing 10 mM Imidazol and 500 mM NaCl, 25 mM Tris-HCl, pH8.

3. Elute protein from Ni column directly to 10 ml MEP column using 50 ml buffer B containing 500 mM NaCl, 250 mM Imidazol, 25 mM Tris-HCl, pH8.

4. Wash column with 100 ml TBS, pH 8, containing 200 mM NaCl.

5. Elute Cre with 50 mM NaOAc buffer, pH5.5, containing 50 mM Arginine, 600 mM NaCl.

2 g NaoAc, 116 ul HoAc, 10 g Arginine, 125 ml 4 M NaCl, H2O to 1L

6. Dialyse eluted protein vs 75 ml of dialysis buffer

50% Glycerol
600 mM NaCl

50 mM Arginine
20 mM HEPES
pH to 7.4

Change buffer  every 4 hours (2 times), then dialyse o/n.

7. Measure protein concentration using Bradford assay or OD280.

8. Add 5, 10, 15, 20 ul into loxP-GFP-loxP-RFP 293 cell line to do activity assay.

9. Aliquote, label  tubes and store at -20 oC o/n

10. Store vials at -80 oC.

Protocol 2,

Based on http://www.jove.com/video/1627/engineering-cell-permeable-protein

1. Inoculate LB/0.5% glucose [g/v]/50 μg/mL carbenicillin with HTNCre glycerol stock using a pipette tip.

2. Grow bacteria at 37°C O/N (for 16 hours).

3. Next day use the densely grown over-night culture to inoculate TB medium with 0.5% glucose, 100 μg/ml Amp (prewarmed to 37 oC) at a ratio of 1:40 or 1:50.

4. Grow bacteria in an incubator at 37°C to OD600 of 1.5.

5. Add 0.5 mM IPTG to induce for 1 h.

6. Spin down bacteria at 5000 rpm for 10 minutes.

7. Store bacteria at -20 oC.

8. Resuspend frozen bacteria pellets in 10 mL lysis buffer per liter flask culture for 15 minutes at room temperature.

9. Add 1 mg/mL lysozyme, incubate at room temperature for 15 minutes while mixing.

10. Add benzonase to 25 U/mL (1:1000) and incubated while mixing for 15 minutes at room temperature.

11. Sonicate on ice for 1.5 min with 0.5 s pulses at 45% of the power.

12. Add 1 mL cold tartaric salt buffer (TSB) per mL suspension while mixing and incubate for 5 min on ice.

13. Take 10 ul lysate for SDS-PAGE analysis (L).

14. Spin at 4°C for 30 min at 12000 RPM. Take 10 ul of supernatant (S) for SDS-PAGE analysis.

15. Transfer supernatnant to fresh 50 mL falcon tubes, add 2 mL of 50% Ni-NTA slurry per liter of initial expression culture, mix gently for 1 h at 4°C in cold room.

16. Pour the suspension into a gravity flow EconoPac column.

17. Take 10 ul flow through for SDS-PAGE analysis (FT).

18. Wash beads twice with 5 bed-volumes of washing buffer. Take 10 ul from washing fractions for SDS-PAGE analysis (W1, W2).

19. Elute HTNCre with 3 bed-volumes of elution buffer, take 10 ul of eluate fraction (E) for SDS-PAGE analysis.

20. Dialyse against High Salt Buffer twice (volume of dialysis buffer to sample is 50), first 1 hour, then O/N at 4 oC.

21. Dialyse against Gylcerol Buffer twice, first 3 ~ 4 hours, then O/N at 4 oC. This will result increase of Cre concentration to 4 folds

22. After dialysis HTCre concentration will be netween 200 and 450 μM, final yield will be ~12 mg per liter of expression culture.

24. Take 1 ul of glycerol stock (GS) for SDS-PAGE analysis.

25. Store HTNCre stock solution at -20°C or -80°C

Material Name	Type	Company	Catalogue Number	Comment
TUNER (DE3) pLacI		Novagen	70625
Glycerol		Carl Roth	3783.2
Na2HPO4		Roth	T876.1
Trizma Base		Sigma-Aldrich	T1503
HCl		Roth	4625.1
Imidazol		Roth	X998.4
NaCl		Roth	9265.2
Yeast Extract		Roth	2363.4
Trypton/Pepton		Roth	8952.4
K2HPO4		Roth	P749.2
KH2PO4		Roth	3904.1
Ampicillin		Sigma	A9518
Carbenicillin		Sigma	6344.2
HEPES		Sigma	H3375
Lysozyme		Sigma	62971
Benzonase		Novagen
L-Tartaric acid, disodium salt		Sigma
50% Ni-NTA slurry		Invitrogen	R901-15
EconoPac columns		Biorad	732-1010
Sterile filter 0,22μm		Whatman
Paraformaldehyde (PFA)		Sigma
LB medium				Yeast extract, Trypton/Pepton, NaCl
TB medium				Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4
Lysis Buffer (PTB)				50 mM Na2HPO4, 5 mM Tris, pH 7.8
Tartaric Salt Buffer (TSB)				PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol
Washing Buffer				PTB, 500 mM NaCl, 15 mM Imidazol
Elution Buffer				PTB, 500 mM NaCl, 250 mM Imidazol
High Salt Buffer				600 mM NaCl, 20 mM HEPES, pH 7.4
Gylcerol Buffer				50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4
TrypLE™ Express		Invitrogen
ESGRO (LIF)		Millipore
NEAA		Gibco	11140035
L-Glutamin		Gibco	25030024
β-Mercaptoethanol		Gibco	31350010
DMEM		Gibco	11960044
PBS		Gibco
Fetal Calf Serum (FCS)		PAA
X-Gal staining solution:				4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS
K3(FeIII(CN)6)		Sigma	P-3367
K4 (FeII(CN)6)		Sigma	P-9387
MgCl2		Sigma	M8266
X-Gal		Sigma	B4252

TB medium 900 ml

Tryptone 12 g
Yeast extract 24g
Glycerol 4 ml
Deionized water or MiliQ water to 900ml

Mix well and autoclave for 20 min at 15 psi on liquid cycle, Store at room temp

Potassium salt 1000 ml

0.17M KH2PO4 and 0.72 M K2HPO4

KH2PO4 23.1g
K2HPO4 125.4g
Deionized water 900 ml

After the salt has completely dissolved, adjust volume to 1000 ml with D.I. water Sterilize by autoclaving for 20 min at 15 psi on liquid cycle
Store at room temp; do not add to TB broth until needed.

Complete TB medium: add 100 ml Potassium salt to 900 ml TB medium.

Protein transduction into murine embryonic stem (ES) cells

1. Seed ES cells carrying a conditional β-Galactosidase reporter construct  as single cells using TrypLE™ Express for dissociation of adherent cells.

2. After 4 to 6 hours the cells had re-attached and remove the medium. (80% confluency)

3. Incubate ES cells with HTNCre-containing medium for 16 hours.

Dilute an appropriate amount of HTNCre protein (corresponding to 10 μM) from the glycerol stock with ES medium and then sterile filtrated (0.22 μm).

4. After protein transduction change medium back to normal growth medium.
5. After two days wash cells with PBS and fixed with 4% Paraformaldehyde (PFA) for 10 minutes.
6. Wash 2 times with PBS

7. Stain fixed cells with X-Gal staining staining solution and incubate over night at 37°

Important: The product we are selling online is Cre from pTriEx-HTNC (His-Tat-NLS-Cre, clone #298) or pET28b-Cre (clone #375). Both of them produce His-Tat-NLS-Cre.

If protein expression level is low, use plasmid to transform Rosetta BL21 (DE3) pLysS completent cells and grow bacterial on plate containing Chl/Car (for pTriEX-HTNC) or Chl/Kan (for pET28b-Cre). Pick a few colonies and grow in 5 ml medium containing Chl/Car (for pTriEX-HTNC) or Chl/Kan for pET28b-Cre), make a glycerol stock, use the rest of cells for protein expression.

Protocol 3 Auto-Induction

1. Day 1: Early Morning: innoculate pTriEx-HTNC (His-Tat-NLS-Cre, clone #298),  pET28b-Cre (His-Tat-NLS-Cre in pET28b  vector, clone # 375),  or pET8XHis-Cre (His-NLS-Cre in pET8XHis, clone # 287) transformed Rosetta BL21 (DE3) pLysS cells (Novagen) O/N in ~ 130 ml LB/Car/Chl (for pTriEx-HTNC) or LB/Kan/Chl (for  pET28b-Cre or pET8XHis-Cre).

2.  At 5 PM: add 20 ml culture to 1 L  LB/Car/Chl or LB/Kan/Chl autoinduce medium. Grow 6 L o/n at 30 oC. Note: set temperature to 20 ~25 ^oC and the incubator will be 30 ^oC. Do not use 37 ^oC.

3. Day 2: In the morning (10 AM to 4 PM, dependent on cell density) : at 6000 rpm for 5 min, wash cells with water and transfer to 50 ml VWR tubes, spin, decant water, add lysis buffer, freezee with dry ice (can store the cell pellet in -80 oC freezer after this step).

IPTG-Induction

1. Day 1: Late Afternoon: innoculate pTriEx-HTNC (6XHis-Tat-NLS-Cre), or  pET28b-Cre, or pET30-8XHis-Cre (His-NLS-Cre) transformed  Rosetta BL21 (DE3) pLysS cells (Novagen) O/N in ~ 250 ml LB-2%Glucose/Car/Chl (for pTriEx-HTNC) or LB-2%Glucose/Kan/Chl/ (for pET28b-Cre or pET30-8XHis-Cre) O/N at 30 oC.

2. Day 2: In the morning, add 40 ml culture to 1 L LB/Car/Chl or LB/Kan/Chl medium. Grow 6 L at 37 ^oC until OD₆₀₀ = 0.6~0.8.

3. Add 0.5 mM IPTG (final conc), induce for 3 ~ 4 hours 30 ^oC. Important: Do not use 37 oC.

4. Collect cells by centrifugation at 6000 rpm for 5 min, wash cells with water and transfer to 50 ml VWR tubes, spin, decant water, add lysis buffer, freezee with dry ice (can store the cell pellet in -80 oC freezer after this step). Continue to Purification step 1 and 2.

Purification

1. Resuspend cell pellets in 30 ml TB per liter flask culture (6L: at least 180 ml), freeze-thaw with dry-ice then 30 oC water for 1 or 2 cycles (when the cells become glue like gel when the cells are lysed)

2. Use homogenizer to break lysed cells (gel solution).

3. Add 20 ul DNase I and let the bottle rock in cold room overnight.

4. Add 3 ml cold TSB to 30 ml suspension.

5. Incubate for 5 min on ice.

6. Spin for 30 min at 12,000 RPM, at 4 oC, Transfer supernatant to clean ultracentrifuge tubes, spin at 30000 rpm at 4 oC for 30 min using ultracentrifuge.

7. Add 10 ml regenerated Ni-NTA beads. Mix by rocking gently in cold room for 60  min to 2 hours.

8. Spin down beads at 6000 rmp for 1 min.

9. Pour supernatant into a clean tube. Be careful not to lose any beads.

10. Pour beads suspension into a gravity flow column, collect flow through, combine flow through and supernatant from step 9.

11. Wash with 5 X 5 bead volumes washing buffer or until no protein is detectable in 10 µl eluate + 75 µl Bradford reagent.

12. Wash beads with washing buffer (50 mM imidazole).

13. Elute HTNCre or His-NLS-Cre with 3-bed volumes (30 ml) of Elution Buffer.

14. Dialyse against 75 ml volumes dialysis buffer or buffer C for 2 hrs. Change buffer every two hours. Repeat 3~4 times.

15. Dialzye against 75 ml glycerol buffer or buffer D to concentrate protein 2 hour each. Repeat 2 to 3 times.

16. Dialzye against 75 ml glycerol buffer or buffer D to concentrate protein o/n.

Concentrate protein by dialyzing against 3 X 75 ml Buffer D ---> 450 uM (corresponds to 19 mg/ml) Cre, change buffer every two hours for the first two dialysis, then overnight for the last dialysis.

17. Transfer dialysed liquid to clean ultracentrifuge tube, spin at 30,000 rpm at 4 ^oC for 20 min.

18. Transfer supernatant to a new 50 ml tube, discard pellet which contains denatured (misfold or inactive) Cre.

19. Make 1:10 serial dilutions of 5 µl protein using Buffer D (not water nor PBS) and measure protein concentration using spectrophotometer and Bradford.

20. Filter through 0.2 µ filter and aliquot into autoclave tubes (1 mg/ml) in the biosafety hood. Avoid forming bubbles.

21. Save a small aliquot for SDS-PAGE analysis to confirm size and purity.

22. Put label (make sure concentration, lot number and size are correct). Upload QC data to ELN. 

23. Put vials in a 96 well paper box,  put the paper box into a styrofoam box (so the protein will be freezed slowing). Store the box in -20 oC o/n, then -80 oC o/N, take out the 96 well box from styroform box and store it at -80 oC.

24. Regenerate Ni beads, add beads to the mixed solution of supernatant from step 9 and flow through from step 10.

25. Repeat step 11 to 20 to purified additional Cre.

This procedure results in glycerol stock solutions containing usually between 200 and 450 micromolar (4~19 mg/ml) HTNCre, at least 2 mg/ml and total 30 mg HTNCre per column purification should be obtained.Usually, 10 to 50 mg of active Cre can be purified from 1 L culture.

Dialysis buffer:

600 mM NaCl
20 mM HEPES
pH to 7.4

Glycerol buffer:

50% Glycerol
600 mM NaCl
20 mM HEPES
pH to 7.4

TB:  Buffer A for PKA alpha purification, i.e., 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 10 mM Imidozale, 10% Glycerol (optional), 0.3 mM EDTA, 2 mM beta-ME.
TSB: tartaric salt buffer, PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM imdazole, Sigma.
Buffer A: PTB, 500 mM NaCl, 15 mM imidazole.

Washing Buffer: TB + 20% glycerol + 20 mM imidazole 

Elution Buffer: 10% glycerol,  250 mM imidazole, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4.
Buffer B: PTB, 500 mM NaCl, 250 mM imidazole.
Buffer C: 600 mM NaCl, 20 mM Tris-HCl, pH 7.4, 20% glycerol.
Buffer D: 50% glycerol, 500 mM NaCl, 20 mM  Tris-HCl, pH 7.4

or 1 X PBS, with additional 250 mM NaCl and 50% glycerol.

Cre Recombination Assay (0.5 to 10 uM Cre is sufficient)

1. Culture CV1-5B and COS-7 cells on a 10 cm2 dish culture plate in normal culture medium for 24 h.
2. Dilute HTNCre glycerol stock solution with culture medium.
3. Filter sterilization with a 0.22 microM filter.
4. Culture cells for indicated time periods in medium containing purified recombinant HTNCre in in a 1:1 mixture of PBS and Glutamax supplemented DMEM (Invitrogen).
* For long-term incubations (over 16 h) , dilute HTNCre to 5 uM with normal FCS-containing medium.
* For growth curve analysis, wash cells were once with PBS after transduction and further cultivate for 5 h in normal medium before being trypsinized and passaged.
* For LacZ reporter gene assays, wash cells after transduction and culture for further 24 h in normal medium before fixation.
* For transduction into ES cells, plate single suspensions of cells in culture medium 6 h prior to Cre transduction, then culture cells for 18 h with recombinant HTNCre in normal ES cell medium. After transduction, wash cells once with PBS and further cultivated for 2 days in normal medium before being further passaged.
* For visualization of HTNCre translocation, incubate CV1-5B cells 30 min with R-HTNCre and wash three times with PBS (Gibco) containing 0.5 mg/ml heparin (Sigma), 5 min each, to remove extracellular bound protein. Use Hoechst 33342 (1 uM) for nuclear counterstaining.

Protocol 4 for high concentration Cre

Immediately after eluting Cre out from column, use Desalting column to desalt (15 ml per injection). Collect and combine enzyme fractions, transfer to 50 ml tubes,  add Ni beads, incubate in cold-room using rotation mixer for 30 min, spin down (1000 rpm, 1 min), decant liquid (do not discard), combine beads, wash with Dialysis buffer, spin, remove as much liquid as possible, add minimal vol of Elution Buffer, soak for 15 min, spin gentle, use pippet to transfer liquid to dialyse tube. Dialyse as step 14 and forward in protocol 1

Protocol 5

Large scale (6L) purification of TAT-Cre (by Jing Wang)

Competent cells: E.coli Tuner (DE3) pLacI (Novagen)
Plasmid: pTriEx (Novagen)-His-TAT-NLS-Cre

PLacI (Novagen) contaning pTriEx -His-TAT-NLS-Cre (Novagen)

1. Inoculate 150ml LB medium. (50ug/mg Carbenicillin---sigma and 34 ug/ml chloramphenicol---sigma in 500ml flask and grow O/N at 37 oC.

2. Remove 1ml LB for OD600 blank. Add 40ml culture to 1L pre-warmed LB (50ug/ml Carbenicillin and 34ug/ml Chloramphenicol) in 2L flasks. Grow at 37C until OD600 is <0.600.

3. Aliquot 100 ml for control.

4. Add 0.5 mM IPTG (2ml, 0.5M stock to each 4L flask) and grow for another 3-4 hrs. Check OD again if possible.

5. Cool down on ice for 10min and harvest in 1L conical flasks and spin 6000 RPM, 4oC for 10 min, aspirate supernatant completely and freeze pellets on dry ice/ethanol both, store at -80C.

Bacterial Lysis

1. Prepare lysis buffer 100ml, take the frozen bacterial on ice and add 10ml to each 1L flask. Wait a few min on ice and resuspend well bacterial pellets, wash with 5ml the same buffer. Transfer to new tubes. Gently mix and incubate 15 min at RT.

2. Add Bugbuster detergent (Novagen#70921), gently mix for 15min RT.

3. Transfer to centrifuge tubes and spin down at 16,000rpm, 4C, 25min.

4. Pool all cleared lysate and filter through 0.22um.
Save 100ul for SDS-PAGE analysis.

Nickel Column

1. Wash Ni-NTA slurry (Qiagen) twice with bead wash buffer (prep. 100ml). resuspend in 1 Vol. bead wash buffer.

2. Save 4ml slurry for pre-loading (2ml/ea.).

3. Divide left-over slurry to 5---50ml tubes. Spin (500rpm, 1min) and aspirate bead wash buffer.

4. Calculate cleared lysate evenly. 1ml packed bead/ 6ml cleared lysate and add to the slurry.

5. Mix gently shaking at 4C at least 1-2hr.

6. Load onto 2 plastic columns (Bio-rad Econo-columns, cat.#737-1512). Preload with the saved 2m l each bead slurry.

7. Let lysate flow through. Save 100ul for SDS-PAGE.

8. Wash column with >10 vol. column wash buffer, O/N Is OK

9. Elute column with elution buffer (100ml), collect 2ml fraction each in cold room.

10. Determine pro-con. By Bradford Assay and pool fractions.

Dialysis

1. Dialyze (10K MWCO, Slidelizer, Perirce) against 100 vol. dialysis buffer >2hrs, repeat 1X or O/N.

2. Dialyze against 4L glycerol buffer to concentrate. Filter through 0.22 mm filter.

Aliquot in hood and slow freeze (-20 C O/N, transfer to -80C).

Clean column

1. wash w/ 2 vol. 6M GuHCl, 0.2 M acetic acid.
2. wash w/ 10 vol dH2O.
3. wash w/ 3 vol 2% SDS.
4. wash w/ 1 vol. 25% EtOH.
5. wash w/ 1 vol. 50% EtOH.
6. wash w/ 1 vol. 75% EtOH.
7. wash w/ 1 vol. 100% EtOH.
8. wash w/ 1 vol. 75% EtOH.
9. wash w/ 1 vol. 50% EtOH.
10. wash w/ 1 vol. 25% EtOH.
11. wash w/ 5 vol. dH2O.
12. wash w/ 5 vol. 0.1MEDTA, pH8.0
13. wash w/ 10 vol dH2O.
14. recharge with 5 vol. 0.1M NiSO4.
15. wash w/ 10 vol dH2O.
transfer to tubes, 4C store.

Solutions:

Stock 1
10X phosphate buffer: 0.5 M NaHPO4 70.89g
50mM Tris 6.06g
adjust pH to 7.8, filter 2L

Stock 2
5M NaCl

Stock 3
1M imidazole adjust pH 7.0, filter

Lysis buffer 100ml

10X phosphate buffer 10ml (stock1)
0.5M NaCl 10ml (stock2)
10 mM imidazole 1ml
1X protease inhibitors (EDTA-free tablets, Roche) 10 tablets
2 mM PMSF
1mg lysozyme/ml 100mg
Benzonase (1:500)
79ml dH2O

Bead wash buffer column wash buffer column elution buffer
(100ml) (1L) (100ml)
Stock1 10ml 100ml 10ml
Stock2 10ml 100ml 10ml
Stock3 1.5ml 20ml 25ml
dH2O 78.5ml 780ml 55ml

phosphate 1x buffer
NaCl 0.5M 0.5M 0.5M
Imidazole 15mM 20mM 250mM

Dialysis buffer:
0.6M NaCl
20 mM HEPES
pH 7.4

Glycerol buffer:
50% Glycerol
0.6M NaCl
20 mM HEPES
pH 7.4
Notes:
1. keep cold as normal protein purification.
2. wear gloves, TAT-Cre can transducer your cells too.

Protocol 6

Large Scale Purification of TAT-Cre (HTNC) Under Native Conditions
Developed by: Michael Peitz(m.peitz@uni-koeln.de), Frank Edenhofer(peitz@uni-bonn.de)
Slightly Modified by: Kevin Otipoby (kotipoby@cbr.med.harvard.edu)

Expression in bacteria
TUNER(DE3)pLacI (Novagen) containing pTriEx(Novagen)-His-TAT-NLS-Cre
1. Inoculate 150 ml LB medium containing 50 ug/m1 Carbenicilli and 34 ug/m1 Chloramphenicol in 500 ml flask and grow overnight at 37°C with shaking.

2. Add 120 ml overnight culture to 6 liters prewarmed LB containing 10011g/m1 Ampicillin (or 50ug/m1 Carbenicillin) and 34ug/m1 Chloramphenicol in three 4 liter flasks.

3. Grow at 37°C with shaking until OD 600 is 0.9-1.0 (approximately 1.5 - 3 hours).

4. Induce expression with 0.5 mM IPTG.

5. Grow cultures for an additional 3 - 4 hours.

6. Cool down flasks quickly in ice bath for 10 min and harvest cells in 6 orange-capped conical flasks (250 ml) by centrifuging at 4200 RPM (Sorvall), 4°C, 20 min. This requires 4 spins; pour off supernatant from each tube and add an additional 250 ml on top of pellet. Once the entire 6 liters have been spun down, aspirate supernatant completely and freeze pellets on dry ice/ethanol bath; store at -80°C.

Bacterial Lysis
7. Thaw and resuspend bacterial pellets in 3 ml resuspension buffer / gram wet weight of bacteria until completely resuspended. Add 1 mg lysozyme/ml suspension.

8. Mix, incubate 20 min at RT.

9. Add 1:1000 Benzonase for additional 15 min at RT.

10. Sonicate 2 min with 0.5 sec pulses on ice.

11. Add 1 ml tartaric salt buffer/ml suspension.

12. Mix gently and incubate for 5 min on ice.

13. Transfer to centrifuge tubes and spin at 16,000 rpm (SS34, Sorvall), 25 min, 4°C.

14. Pool cleared supernatant. Filter through 0.22 um filter (this step takes a while and probably requires multiple changes of filters).

Nickel Column Purification

15. Wash Ni-NTA slurry (Qiagen) twice with bead wash buffer. Resuspend packed beads in 1 volume bead wash buffer.

16. Add 2 ml bead slurry (1 ml packed beads) / 6 ml cleared lysate and mix by gently shaking at 4°C for 1 hr.

17. Load onto 2 plastic columns (Bio-Rad Econo-columns, cat. #737-1512) preloaded with 2 ml bead slurry (1 ml packed beads).

18. Let lysate flow through.

19. Wash with at least 10 bed volumes wash buffer.

20. Elute with elution buffer collecting 2 ml fractions.

21. Determine which fractions contain protein by Bradford Assay and pool protein-containing fractions. If solution gets turbid, add more elution buffer until protein is dissolved.

22. Determine protein concentration by Bradford Assay with BSA standard and (optional) endotoxin contamination by LAL Assay.

Dialysis
23. Dialyze (10K MWCO, Slidelizer, Peirce) against 100 volumes dialysis buffer 2 hrs (minimum). Repeat 1x.

24. Dialzye against 1 liter glycerol buffer to concentrate.

25. Filter through 0.22 um filter, aliquot in sterile tubes.

26. Freeze (place the vials in a Styrofoam container in the —20° C overnight), then Transfer to —80°C freezer.

Solutions
10x phosphate buffer:
0.5 M Na2HPO4
50 mM Tris adjust pH to 7.8, sterile filter

imidazole stock

1 M imidazol
adjust to pH 7.0 (with conc. HO), sterile filter

resuspension buffer:
1 x phosphate buffer
500 mM NaCl
10 mM imidazole
0.5x protease inhibitors (EDTA-free tablets, Roche)

Lysis enzymes

rlysozyme ( ovagen)
Benzona (Novagen)

bead wash buffer
1x phosphate buffer
20mM imidazole
500 mM NaCl

tartaric salt buffer:
2M L-Tartaric acid (Disodium salt)
lx phosphate buffer
15 mM imidazole

column wash buffer:
1x phosphate buffer
500 mM NaCl
20 mM imidazole

column elution buffer:
1x phosphate buffer
500 mM NaCl
250 mM imidazole
pH to 7.4

glycerol buffer:
50% Glycerol
600 mM NaC1
20 mM HEPES
pH to 7.4

Dialysis buffer
600 mM NaC1
20 mM HEPES
pH to 7.4

Basic protocol for TAT-Cre transduction

Harvest organs and purify cells as normal.

Wash cells 3x in Hyclone ADCF-Mab serum-free media. If you do not wash 3x, your efficiencies will be lower (probably about 25-35%).
Resuspend cells at 107/m1 in Hyclone serum-free media. Dilute TAT-Cre to 100 pz/ml in Hyclone serum-free media. Prewarm to 37 degrees C for about 10 min.
Add 1:1 cells and TAT-Cre (or Hyclone media for a —TAT-Cre control). Final conc of cells = 5x106 cells/ml
Final cone of TAT-Cre = 50 1.1g/m1 (1.25 11M)
Incubate 45 min at 37 degrees C.
Stop transduction with media containing 10% FCS. If the volume of cells and TAT-Cre is large, I add FCS to final conc of 10%, then fill the tube with media containing 10% FCS.
Spin, wash cells with media containing 10% FCS. Place in culture.

Notes:
1) Wear gloves when handling TAT-Cre; it will transduce your skin cells as well as lymphocytes.
2) Transduction efficiency should be around 45-55% in primary B cells.
3) There is some toxicity associated with TAT-Cre transduction. You should recover at least 75-80% of the cells. If you go up on the dose of TAT-Cre or longer on the time of incubation, the transduction efficiency does not go up but the recovery goes down. This might not be the case for cells other than primary B cells, though.
3) Transduction in the presence of synthetic TAT-HA (see Wadia et at., Nature Medicine. 2004) allows one to use TAT-Cre at lower doses which gives near 100% recovery of cells.
4) You can transduce cells in the presence of FCS, but you have to use much more TAT-Cre.

5), opti-MEM can be used to replace Hyclone ADCF-Mab serum-free media

Reference

for construction of HTNC plasmid: Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F. 2002 PNAS 99(7):4489-94.
Other relevant references for TAT-Cre:

Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE. 2001 Nature Biotechnology19(10):929-33.
Joshi SK, Hashimoto K, Koni PA. 2002 Genesis 33(1):48-54. Wadia JS, Stan RV. Dowdy SF. 2004 Nature Med 10

Cre activity assay

Cre purified with

• 1. Ni column only: High activity, high toxicity
• 2. Ni column then SP: medium activity, medium toxicity
• 3. Ni column then MEP: very low activity, low toxicity.Cre Expression/Induction using Auto-Induce, IPTG and L-arabinose

Clone 1708, and 1010

Load Cre from 1010 to 20 ml QFF-->50 SP-FF, elute with 300 ml 0f 100 mM-->1M NaCl linear gradient 

Expression screening using 10 ul magnetic beads from expedeon Amintra® Nickel Magnetic Affinity Resin

Lane	ID	Name	Host	MW	Induction	Note	Expression
1	375	pET28-Cre	Nico21(DE3)pLysN,	10.30 / 42.8	1 mM IPTG, 18 oC	grown on LB/KC, most cells are lysed	y
2	375	pET28-Cre	Rosetta(DE3)	10.30 / 42.8	1 mM IPTG	looks like contaminated, can not lyse cells	trace
3	375	pET28-Cre	Nico21(DE3)pLysN	10.30 / 42.8	1 mM IPTG	grown on LB/K	y
4	2603	pL6H-sso7d-SD-Taq	Nico21(DE3)pLysN	6.10 / 104.7	1 mM IPTG		y
5	2642	pET29b-Flp	Nico21(DE3)pLysN	9.76 / 52.5	1 mM IPTG	most cells are lysed	n
M			pageruler plus
6	2643	pET29b-Int-C3	Nico21(DE3)pLysN	9.69 / 41.4	1 mM IPTG		maybe
7	2643	pET29b-Int-C3	Nico21(DE3)pLysN	9.69 / 41.4	1 mM IPTG		maybe
8	2644	pET29b-scIHF2	Nico21(DE3)pLysN	9.60 / 22.8	1 mM IPTG		?
9	1010	Cre	Nico21(DE3)pLysN			Lot 174-1010
10	1186	MBP or MBP-Xis

Purification of Cre using Ni-NTA mag beads from Genscript and Ni_NTA agarose from GE

Lane	Sample
1	Ni-NTA Mag beads sample 1 frac2
2	Ni-NTA Mag beads sample 1 frac3
3	Ni-NTA Mag beads sample 1 frac4
4	Ni-NTA Mag beads sample 2 frac2
5	Ni-NTA Mag beads sample 2 frac3
6	Ni agarose column  16mm    frac 1
7	Ni agarose column  16mm    frac 2
8	Ni agarose column  2.5 cm  frac 8
9	Ni agarose column  2.5 cm  frac 11
10	Ni agarose column  2.5 cm  frac 14
11	Ni agarose column  2.5 cm  frac 17
M	pageRuler Plus