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

== <br/>Protocol 6 ==

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

'''Expression in bacteria '''<br/>TUNER(DE3)pLacI (Novagen) containing pTriEx(Novagen)-His-TAT-NLS-Cre<br/>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'''<br/>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.

<br/>'''Dialysis'''<br/>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'''<br/><u>10x phosphate buffer:</u><br/>0.5 M Na2HPO4<br/>50 mM Tris adjust pH to 7.8, sterile filter

<u>imidazole stock</u>

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

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

<u>Lysis enzymes</u>

rlysozyme ( ovagen)<br/>Benzona (Novagen)

<u>bead wash buffer</u><br/>1x phosphate buffer<br/>20mM imidazole<br/>500 mM NaCl

<u>tartaric salt buffer:</u><br/>2M L-Tartaric acid (Disodium salt)<br/>lx phosphate buffer<br/>15 mM imidazole

<u>column wash buffer: </u><br/>1x phosphate buffer<br/>500 mM NaCl<br/>20 mM imidazole

<u>column elution buffer:</u><br/>1x phosphate buffer<br/>500 mM NaCl<br/>250 mM imidazole<br/>pH to 7.4

<u>glycerol buffer:</u><br/>50% Glycerol<br/>600 mM NaC1<br/>20 mM HEPES<br/>pH to 7.4

<u>Dialysis buffer</u><br/>600 mM NaC1<br/>20 mM HEPES<br/>pH to 7.4

<br/><u>Basic protocol for TAT-Cre transduction</u>

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%).<br/>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.<br/>Add 1:1 cells and TAT-Cre (or Hyclone media for a —TAT-Cre control). Final conc of cells = 5x106 cells/ml<br/>Final cone of TAT-Cre = 50 1.1g/m1 (1.25 11M)<br/>Incubate 45 min at 37 degrees C.<br/>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.<br/>Spin, wash cells with media containing 10% FCS. Place in culture.

<u>Notes:</u><br/>1) Wear gloves when handling TAT-Cre; it will transduce your skin cells as well as lymphocytes.<br/>2) Transduction efficiency should be around 45-55% in primary B cells.<br/>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.<br/>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.<br/>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



<u>Reference</u>

for construction of HTNC plasmid: Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F. 2002 PNAS 99(7):4489-94.<br/>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.<br/>Joshi SK, Hashimoto K, Koni PA. 2002 Genesis 33(1):48-54. Wadia JS, Stan RV. Dowdy SF. 2004 Nature Med 10