Restriction Enzyme Purification

Kpn1

1. Suspend cells in Buffer A 10 mM potassium phosphate (pH 7·0), 1 mM EDTA, 7 mM b-mercaptoethanol. and lyse cells by freezing-thawing and homogenization.

2. Spin at 25000 rpm for 30 min.

3. Collect supernatant.

Optional add 1% polyethyleneimine (PEI) or freshly made 10% (w/v) streptomycin sulfate was added to a final concentration of 1 %.
in the presence of 250 mM NaCl. Spin again for 15 min to precipitate DNA. Then add ammonium sulphate to 50% for fractionation. Spin to and collect pellet. Dissolve pellet in 5 ml Buffer A and dialyze against buffer A o/n.

4. Load sample to a 5 ml Hi-Trap heparin column or large scale 70 ml  column volume, wash and elute the enzyme with Buffer A containing 2–0·8 M NaCl. Combine the fractions containing the enzyme (between 200–350 mM NaCl), dialyse against Buffer B 20 mM Tris-HCl, pH 7·4, 0·1 mM EDTA, 50 mM NaCl, 5 mM b-mercaptoethanol.

5. Load to Mono-Q (HR1O/10) column and eluted with a linear gradient from 0.1 M to 0.5 M NaCl in Buffer B.

6. Pool fractions containing Kpn1 and dialyse against storage buffer, 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.2 M KCl, 1 mM DTT, and 50 % glycerol.

50 milligrams of KpnI can be purified from 12 g of E. coli AH29. The molecular weight (Mr) of KpnI is
approximately 31,000 daltons.

Not1

Grow E. coli ER2169 (NEB# 816) in LB +0.2% Glucose /Amp/Chl to log phase at 37 oC. Add  IPTG to a final concentration of 0.5 mM.

Induce for 2 hours at 37 oC.

Spin down cells, and freeze at -80 oC.

All the following procedures should be performed either on ice or at 4° C.
1. Resuspend 18 grams of cells in 70 ml of Buffer A

(10 mM potassium phosphate pH 6.9, 0.1M NaCl, 0.1 mM EDTA, 5% glycerol)

2. Break cells by freeing-thawing and then homogenization.

3. Spin at 25000 rpm for 30 min at 4° C.

4. Load the resulting supernatant onto a phosphocellulose column (2.5×15 cm) equilibrated with buffer A.

5. Wash the column with 60 ml buffer A and then a 900 ml linear gradient from 0.1M NaCl to 1.0M NaCl.

enzyme should be eluted at 0.2 to 0.3M NaCl

6. Pool fractions, concentrate and dialyze against buffer B (0.1M NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5% glycerol and 0.15% NaAzide) overnight, or buffer exchange using Sephadex G25.

7. Load onto a Heparin-Sepharose column (1.5×17 cm) equilibrated with buffer B.

8. Wash the column with 30 ml buffer B, and a 400 ml linear gradient from 0.1M NaCl to 1.2M NaCl in Buffer B.

The restriction enzyme should be eluted from heparin column between 0.5M and 0.6M NaCl, pool fractions.

9. Dilute the pooled solution 1:1 with buffer B without NaCl.

10.Load to a DEAE-Sepharose column (1.5×16 cm) equilibrated with buffer B at 0.3M NaCl.

The enzyme will not bind and thus should flow through the column.

11. Collect flow-through diluted 1:1 with buffer B no NaCl, and loaded onto a PEI column (1.5×10 cm) equilibrated with buffer B at 0.15M NaCl.

12. Wash the column with 20 ml buffer B, and a 300 ml linear gradient from 0.15M to 1.8M NaCl.

13. The enzyme should be eluted at 0.6M to 0.8M NaCl.

14. Add BSA (bovine serum albumin) to the pool to a final concentration of 100 μg/ml.

15. Dialyse the enzyme against storage buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1M NaCl, 1 mM DTT, 0.15% Triton-X 100 and 50% v/v glycerol).

This purification scheme should yield 53,000,000 units of NotI restriction endonuclease

QC

The NotI restriction endonuclease obtained from this purification method should be substantially pure and free of non-specific endonuclease and exonuclease.

1), Ligation: After a 25-fold overdigestion of Adeno2 DNA, greater than 95% of the DNA fragments produced can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 uM at 16° C.). Of these ligated fragments, 95% should be able to be recut.

2), Prolonged digestion: After incubating a 50μl reaction containing 1 μg of Adeno2 DNA and 200 units of enzyme for 16 hours, the same pattern of DNA bands should be produced as a reaction performed in one hour with one unit of enzyme.

3), Exonuclease Activity: After incubation of 200 units of enzyme for 4 hours at 37° C. in a 50μl reaction containing 1 μg sonicated 3 H DNA (105 cpm/μg) less than 0.03% radioactivity should be released.

4), Endonuclease Contamination: After incubation of 200 units of enzyme for 4 hours at 37° C. in a 50μl reaction containing 1 μg φX174 RFI DNA, less than 5% should be converted to RF II.

5), Megabase genomic Digest: Digestion of one microgram of E. coli genomic DNA embedded in 0.5% agarose incubated with 200 u NotI for 16 hours in 100μl NEBuffer#3 resulted in the same limit digest banding pattern as 5 u in 4 hours, as determined by PFG electrophoresis.

All tests can be performed in the following reaction buffer:

NEBuffer 3 (100 mM NaCl , 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.9 at 25° C.) supplemented with 100 μg/ml BSA.

References:

EcoRII (Kosykh VB et al. (1980) Mol. Gen. Genet. 178:717-718), EcoRI (Newman AK et al. J. Biol. Chem. 256:2131-2139 (1981), and Greene PJ et al., J. Biol. Chem. 256:2143-2153 (1981)), EcoRV (Bougueleret L et al. Nucl. Acids Res. 12:3659-3676 (1984)), PvuII (Blumenthal RM et al. J. Bacteriol. 164:501-509 (1985)), and PaeR71 (Gingeras TR and Brooks JE Proc. Natl. Acad. Sci. USA 80:402-406 (1983))

DdeI (Howard KA et al. Nucl. Acids Res. 14:7939-7950 (1989)), BamHI (Brooks JE et al. Nucl. Acids Res. 17:979-997 (1989))