Preparing bacterial cultures for protein production

1. Prepare antibiotic solutions:
2. 10 mg/ml tetracycline (in 50% EtOH)
3. 100 mg/ml ampicillin
4. 34 mg/ml chloramphenicol (in 100% EtOH)
5. 0.1M ZnCl (supplement)
   1. Start O/N culture of LB with 1/1000 of each antibiotic/supplement. For each 1L flask , do a start-up culture of 50 ml.
   2. Grow on shaker (37 degrees C, 200 rpm) O/N.
   3. Mix 1L LB media with 1ml (1/1000 of total volume) antibiotics/supplement. Add the 50 ml of O/N culture , take 1ml aliquot for OD measurement *, and put 1L culture on shaker (37 degrees C, 200rpm).
   4. *The OD is measured at 600nm (with LB as blank).
   5. When OD has reached 0.5 (it takes 2-3 hrs.), transfer flasks to a room temperature (RT) shaker and ahake for an hour.
   6. Remove 1ml from each flask for the pre-induction sample. Measure the OD. Add a 1/1000 dilution of 0.7M IPTG (667mg IPTG/4mls ddH2O, prepared fresh ) to each flask and begin induction. Pellet the pre-induction sample by spinning 90 sec. And store pellets at -20 degrees C.
   7. Continue shaking for ~ 3-4 hrs (at RT). Again remove 1ml from each flask, for post-induction sample. Measure the final OD. Pellet the post-induction sample. Spin and store the pellets.
   8. Spin down cells 20 min. at 4000 rpm (at 4 degrees C), using large swing bucket rotor (JS5.2). Decant fluid except for ~30-40ml. Resuspend cells in this liquid and transfer to 50 ml conical tubes and repellent as above. Drain remaining culture fluid and freeze in liquid nitrogen. Store at -80 degrees C.
   9. Run a 15% SDS-PAGE gel on the pre-induction and post-induction samples to determine amount of total protein produced and amount of insoluble and soluble proteins.

SDS-PAGE to test Degree of Induction (Protein Production)

1. •  Add 20mM TrisCl ph 7.9
2. •  400 ul to pre-induced pellet, vortex, then transfer 8ul of sample to new tube.
3. •  200 ul to post-induced pellet and vortex.
4. •  Sonicate resuspended post-induced pellet
5. •  2 x 20 pulses, tip @ 5, 50% duty.
6. •  Vortex sample and remove 8 ul total sample (to new tube).
7. •  Spin remaining material for 5 min. at full speed (14000rpm) and transfer supernatant to new tube. Remove 8ul for soluble sample .
8. •  Resuspend pellet in 200ul of TrisCl pH 7.8. Remove 8 ul for pellet sample .
9. •  Add 4ul of load buffer to all samples: pre-induced, total, supernatant and pellet.
10. •  Boil all samples for 5 min. and quickly spin down material.
11. •  Load onto a 15% SDS-PAGE gel (15 well comb) and run gel @ 30mA (usually takes ~ 45 min). If you're running 2 gels, run at 60 mA, or you're going to need to run for 90 min, instead of 45 min.
12. •  Stain gel for 30 min.
13. •  Destain O/N.

 

Purification of Chimeric Nucleases

Buffers for Chimeric nuclease purification

 

Binding buffer

200 ml 500 ml

End concentration (after mix) Stock concentrations

• H 2 O 134.6 ml 336.5 ml
• 20 mM Tris-HCl pH 7.9 4 ml 10 ml 1 M Tris
• 5 mM imidazole pH 7.9 1 ml 2.5 ml 1 M imidazole
• 500 mM NaCl 20 ml 50 ml 5 M NaCl
• 10 % glycerol 40 ml 100 ml 50 % glycerol
• 0.1 mM ZnCl 2 0.2 ml 0.5 ml 0.1 M ZnCl 2
• 0.1 mM PMSF 0.2 ml 0.5 ml 0.1 M PMSF

60 mM imidazole Wash 400 mM imidazole Elute

50 ml 50 ml

• H 2 O 30.9 ml 13.9 ml
• 20 mM Tris-HCl pH 7.9 1 ml 1 ml 1 M Tris
• Imidazole 3 ml 20 ml 1 M imidazole
• 500 mM NaCl 5 ml 5 ml 5 M NaCl
• 10 % glycerol 10 ml 10 ml 50 % glycerol
• 0.1 mM ZnCl 2 50 m l 50 m l 0.1 M ZnCl 2
• 0.1 mM PMSF 50 m l 50 m l 0.1 M PMSF

TDZ buffer

Basic 400 mM NaCl 1M NaCl

100 ml 10 ml 10 ml

• H 2 O 78 ml 7.2 ml 6 ml H 2 O
• 20 mM Tris 2 ml 0.2 ml 0.2 ml 1 M Tris
• 10 % glycerol 20 ml 2 ml 2 ml 50 % glycerol
• NaCl --- 0.8 ml 2 ml 5 M NaCl
• 10 mM b -mer 78 m l 8 m l 8 m l b -ME
• 0.1 mM ZnCl 2 100 m l 10 m l 10 m l 0.1 M ZnCl 2
• 0.1 mM PMSF 100 m l 10 m l 10 m l 0.1 M PMSF

Purification of Chimeric Nucleases

 

First column (Ni-NTA) Run

1. Cells frozen at -80°C are thawed at room temperature.
2. While the cells are thawing, begin preparing the His-bind affinity column by packing it with ~ 2ml of Ni-NTA resin (conc. of actual resin is ~50% in suspension, so use 4ml of suspension).
3. Continue with by washing: first with 10 volumes (i.e 20ml) of ddH2O, followed by 5 volumes of charge buffer (50 mM NiSO4 , and 3-4 volumes of binding buffer. We use a pump set at 3 or higher speed. Monitor the column occasionally so that the column doesn't dry out or you'll have to repack it. Try to use a speed that will maintain the liquid level(head volume) above the top of the resin throughout the whole purification.
4. Thawed cells preparation:
   1. The thawed cells are resuspended in 40 ml of binding buffer(20 mM Tris-CI pH 7.9, 5 mM imidazole, 100 uM ZnCI2, 10% glycerol, 500 mM NaCI, and 0.1 mM PMSF/pefablock ), transferred to a 100 ml plastic beaker, and stirred for about 15 minutes at room temperature.
   2. Add 200 ul of 20% TritonX-100, dropwise, to cell mixture, and stir further for about 10 minutes. You want about a 0.1% final concentration of Triton. The cell suspension should become more viscous and transparent.
   3. After stirring, place the beaker on ice and sonicate. Sonicate for 1 minute, 3-SXon duty 50% and power of 6. Interrupt sonication with 1 minute intervals on ice to prevent excessive warming of the tip and cells.
   4. Centrifuge the material in 50 ml polypropylene oakxidge tubes at 18,000 rpm in a JA-20 rotor for 1 hour.
   5. After centrifugation, the crude extract is filtered through 0.45 uM filter(s). The supernatant** is loaded Onto a His-bind affinity column at a speed of 2 or 3. Collect an aliquot of the flow-through**.
5. After loading, which takes around 3 hrs, the column is washed using a pump speed of 3, first with 10 volumes of binding buffer followed by 6 volumes of binding buffer with 60 mM imidazole. Remove an aliquot of the second wash.**
6. The protein is eluted with binding buffer containing 400 mM imidazole using a speed of 2. The protein is collected with a fraction collector into 1.7 ml microfuge tubes (0.5 ml/fraction), it is usually eluted within 4 volumes.
7. Test the fractions with a crude Bradford assay( 5 ul of fraction ± 50 ul of Bradford rgt. solution).
8. **50 ul aliquots are removed at these times during the purification: supematant, flow-through, wash, Ni~ fractions, pooled Ni fractions, flow-through, wash, Heparin fractions.
9. If you have time, you can further purify the fractions using a Heparin-Sepharose column. Otherwise, the fractions can be frozen in liquid nitrogen and stored at -80°C

Second column (Heparin-Sepharose) Run

1. The Heparin-Sepharose column is prepared fresh each time with a 0.8 ml bed volume.
2. The column is washed by gravity with ddH2O.
3. This is followed by washing with TDZ buffer 2O mM Ths-C1 pH 7.9, 100 uM ZnCL, 10 mM BME, 0.1 mM PMSF on a speed of 3 or higher.
4. 50 ul aliquots are removed from the fractions containing the most protein based on the Bradford assay performed after the His-bind column. The protein containing fractions are combined, diluted 2.5x with TDZ buffer. The buffer is added very slowly and mixed very gently to prevent precipitation of the protein. Remove another aliquot of the "pooled" fractionS.**
5. The diluted fractions are loaded**, then washed with 6 volumes of TDZ buffer with 400 mM NaCl**.
6. The material is eluted with 4 volumes of TDZ with 1 M NaCI. Fractions are collected with a fraction collector, again on a speed of 2
7. The fractions are again analyzed by a crude Bradford assay.
8. An aliquot** of the best fractions are run on a 15% SDS-PAGE gel to determine purity.

**50 ul aliquots are removed at these times during the purification: supematant, flow-through, wash, Ni~ fractions, pooled Ni fractions, flow-through, wash, Heparin fractions.