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

== Protocol 3 Auto-Induction ==

1. Day 1: Early Morning: innoculate pTriEx-HTNC (His-Tat-NLS-Cre, clone #298),&nbsp; pET28b-Cre (His-Tat-NLS-Cre in pET28b&nbsp; vector, clone # 375),&nbsp; 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&nbsp; pET28b-Cre or pET8XHis-Cre).



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



3. Day 2: In the morning (10 AM to 4 PM, dependent on cell density)&nbsp;: 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&nbsp; pET28b-Cre, or pET30-8XHis-Cre (His-NLS-Cre) transformed&nbsp; 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 <sup>o</sup>C until OD<sub>600</sub> = 0.6~0.8.

3. Add 0.5 mM IPTG (final conc), induce for 3 ~ 4 hours 30 <sup>o</sup>C. '''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'''

<br/>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&nbsp; 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&nbsp;His-NLS-Cre with 3-bed volumes (30 ml) of&nbsp;'''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&nbsp;or buffer D''' to concentrate protein o/n.

Concentrate protein by dialyzing against 3 X 75 ml Buffer D --->&nbsp;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 <sup>o</sup>C 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.&nbsp;

23. Put vials in a 96 well paper box,&nbsp; 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<br/>20 mM HEPES<br/>pH to 7.4

'''Glycerol buffer''':

50% Glycerol<br/>600 mM NaCl<br/>20 mM HEPES<br/>pH to 7.4

'''TB:''' &nbsp;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.<br/>'''TSB:''' tartaric salt buffer, PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM imdazole, Sigma.<br/>'''Buffer A''': PTB, 500 mM NaCl, 15 mM imidazole.

'''Washing Buffer:&nbsp;'''TB + 20% glycerol + 20 mM imidazole&nbsp;

'''Elution Buffer: '''10% glycerol,&nbsp; 250 mM imidazole, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4.<br/>'''Buffer B''': PTB, 500 mM NaCl, 250 mM imidazole.<br/>'''Buffer C:''' 600 mM NaCl, 20 mM Tris-HCl, pH 7.4, 20% glycerol.<br/>'''Buffer D''': 50% glycerol, 500 mM NaCl, 20 mM&nbsp; 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.<br/>2. Dilute HTNCre glycerol stock solution with culture medium.<br/>3. Filter sterilization with a 0.22 microM filter.<br/>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).<br/>* For long-term incubations (over 16 h) , dilute HTNCre to 5 uM with normal FCS-containing medium.<br/>* 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.<br/>* For LacZ reporter gene assays, wash cells after transduction and culture for further 24 h in normal medium before fixation.<br/>* 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.<br/>* 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.