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

== '''Protocol 2''' ==

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



'''Note:'''

*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.



<br/>'''Buffers'''<br/><u>Buffer A</u>, 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.<br/><u>TT buffer</u>: 500 mM disodium tartrate.<br/><u>Ni Washing buffer</u>: the same as Buffer A.<br/><u>Ni Elution buffer</u>:&nbsp; Buffer A with 250 mM imidazole. Store at 4 oC.

<u>MEP Equilibration Buffer</u>: 20 mM&nbsp; 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.

<u>MEP Washing Buffer:</u> 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.



<u>MEP Elution Buffer:</u> <strike>20 mM Phosphate Buffer, pH 6.0</strike>, 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.



<u>SP Equilibration Buffer:</u> 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.





<u>SP Washing Buffer:</u> 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.

<u>SP Washing Buffer II</u>: '''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.



<u>SP Elution Buffer</u>: 20 mM Phosphate Buffer, pH 7.4, 1 M NaCl,&nbsp; 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.

<br/><u>Dialysis Buffer I</u>: 500 mM NaCl, 20 mM&nbsp; Phosphate Buffer, pH 7.4, 50 mM Arginine.<br/><u>Dialysis Buffer II</u>: 50% glycerol, 500 mM NaCl, 20 mM Phosphate Buffer,&nbsp; pH 7.4,&nbsp;50 mM Arginine.

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

'''Expression and Purification'''

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

<strike>pET28bCre: C43(DE3) pLysS or Nico(DE3)/pLysN</strike>



<strike>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'''</strike>

3. Inoculate 200 ml LB medium supplemented with <strike>1% (w/v) glucose</strike>, 50 ug/ml <strike>'''carbenicillin''', </strike>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''', <strike>100 ug/ml '''ampicillin'''</strike>, 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 <strike>0.5 mM (add 0.5 ml of 1 M IPTG to 1 L</strike>) and continue incubation for <strike>1 to 2 hours</strike>

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

For '''Nico(DE3)/pLysN''': induce with 0.1 mM IPTG o/n at 25 oC, or 6 hour at 30 oC.

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



7. Thaw pellets in warm water and resuspend in&nbsp; '''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.

<br/>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).

<br/>11. Centrifuge the lysate for 30 min at 4 oC and&nbsp; 12000 rpm (do not run > 12000 rpm).

<u>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.</u>

<br/>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.&nbsp;

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

14. Equilibrate 5 (or 10) ml MEP column with <u>MEP Equilibration Buffer</u>.

15. Elution of the protein directly to the MEP column with 10 ~15 CV&nbsp; <u>MEP Elution Buffer</u>,

16. Wash column with at least 10 to 15&nbsp; CV of <u>MEP Washing Buffer&nbsp; </u>until no protein is detectable.

17. Elute Cre protein directly to 5 (or 10&nbsp; ml) of SP or CM column with 10 to 15 CV of <u>MEP Elution Buffer.</u> Close the column valve.

Note: <strike>add 1 vol of dilution buffer (20 mM Phosphate Buffer, pH 7.5) to eluate if elution to CM column.</strike> 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 <strike>or CM resin.</strike>

20. Wash SP <strike>or CM</strike> column with 4 CV of&nbsp; <strike><u>CM Washing Buffer</u> or</strike> <u>SP Washing Buffer.</u>

21. Elute Cre from SP <strike>or CM column</strike> with 5 to 10 CV of&nbsp; <strike><u>CM Elution Buffer or</u></strike><u>SP Elution Buffer</u>, 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 <strike>CM or</strike> 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.&nbsp; 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 '''<u>Dialysis Buffer I</u>&nbsp; '''(in 100 ml beaker) for 3~4 hour in cold room.&nbsp; Measure OD280. Dialyze Cre protein again for 3~4 h in cold room. Measure OD280.

28. Dialyse with 50 ml of <u>'''Dialysis Buffer II'''</u> 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.&nbsp;&nbsp; Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2 mg/vial. &nbsp; The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

31. Put vials immediately on ice.&nbsp; Make sure the protein is '''at least 1 mg per vial''' (give 1.2 mg so customers will not complain). Create a new lot number in the product database, make sure to record all product information including&nbsp; 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 <sup>o</sup>C freezer. After 12 hours, take the foam box out and store the freezer box in -80 <sup>o</sup>C freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.



Cre protein sequence:

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

<br/>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.



=== <br/>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&nbsp;: F12 medium, then add again the suitable culture medium.

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