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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of Bacterially Produced His-tagged Proteins on a Nickel Column

Purification of Bacterially Produced His-tagged Proteins on a Nickel Column
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
This protocol describes the purification of a bacterially expressed protein containing 6 tandem histidine residues. The fusion protein can be readily purified on a nickel column owing to the remarkable affinity of the (6X)His tag to the divalent nickel cations.
 
Procedure
A. Cell Growth

1. Inoculate a 100 ml culture of 2X YT + 0.04% Glucose and the appropriate antibiotic with 0.5 ml of an overnight culture of bacteria transformed with the plasmid carrying the gene encoding the His-tagged protein of interest.

2. Incubate cells in growth media at 37°C with shaking until the Optical Density at 600 nm (OD600) is between 0.6 to 1.0 (approximately 3 hours).

3. Induce expression of the heterologous protein by addition of IPTG to 3 mM final concentration (see Hint #1).

4. Continue incubation for 3 to 5 hours.

B. Preparation of Bacterial Extract

1. Harvest the cells by centrifugation at 4°C for 20 min at 3,900 X g (4,900 rpm using a GSA rotor).

2. Discard the media and weigh the cell pellet.

3. Resuspend in Manley Buffer D at 2 ml/gram of cell pellet OR 0.75 ml per initial 100 ml of culture.

4. Sonicate cells two times for 15 sec each.

5. Centrifuge in a microcentrifuge at maximum speed to pellet the cell debris.

6. Discard the supernatant.

7. Resuspend pellet in 1 ml of Guanidine Solution, pH 7.9 (see Hint #2).

8. Gently shake the suspension at 4°C for 40 min to 1 hour.

9. Centrifuge in a microcentrifuge at maximum speed to pellet the cell debris.

10. Save the supernatant.

11. Centrifuge the supernatant in a microcentrifuge for 10 seconds at maximum speed to pellet the cell debris.

12. Save the supernatant.

C. Purification Over Nickel Affinity Column

1. Prepare a 0.5 ml nickel (2+)-Nitrilotriacetic Acid (Ni-NTA) affinity column (follow the manufacturer's instructions).

2. Wash the column with 5 column volumes (2.5 ml) of Guanidine Solution, pH 7.9.

3. Load the supernatant from Step B10 onto the washed column, collect the flow thru, and pass the collected flow thru over the column again. Repeat this step one more time so that the supernatant has passed over the column a total of 3 times.

4. Wash the column with 10 column volumes of Guanidine Solution, pH 7.9.

5. Wash the column with 10 column volumes of Guanidine Solution, pH 6.0.

6. Elute the bound protein with 2 ml of Guanidine Solution, pH 5.0.

7. Collect 0.5 ml fractions from the column.

8. Analyze an aliquot of each fraction by SDS-PAGE (see Protocol ID#455).

9. Pool peak fractions containing the protein of interest (usually around fraction #2 and #3).

10. Dialyze pooled fraction against Guanidine Dialysis Buffer #1 for at least 1.5 to 2 hours at 4°C.

11. Dialyze pooled fraction against Guanidine Dialysis Buffer #2 for at least 1.5 to 2 hours at 4°C.

12. Prepare aliquots of the dialyzed protein and snap freeze in Liquid Nitrogen (see Hint #3).

13. Store purified His-tagged protein aliquots at -80°C.

14. Wash the column with 5 column volumes of Guanidine Solution, pH 2.0. Then wash the column with 5 column volumes of Guanidine Solution, pH 7.9 to regenerate the column for future use.

Solutions
2X YT Media   5 g NaCl
16 g Tryptone
Autoclave
Adjust pH to 7.0 with 5 M NaOH
10 g Yeast Extract
Guanidine Dialysis Buffer #2   0.5 M Guanidine
Prepared in Manley Buffer D
Guanidine Dialysis Buffer #1   2 M Guanidine
Prepared in Manley Buffer D
Guanidine Solution, pH 2.0   20 mM Glycine, pH 2.0
6 M Guanidine
Guanidine Solution, pH 5.0   6 M Guanidine
2 mM Sodium Acetate, pH 5.0
Guanidine Solution, pH 6.0   20 mM PIPES, pH 6.0
6 M guanidine
Guanidine Solution, pH 7.9   80 mM HEPES, pH 7.9
6 M Guanidine
Manley Buffer D   15% (v/v) Glycerol
20 mM HEPES-KOH, pH 7.9
0.2 mM EDTA
42 mM (NH4)2SO4
Add DTT just before use
0.5 mM DTT
IPTG   0.1 M Isopropylthio-β-D-galactoside (IPTG)
Store at -20°C
Filter-sterilize
10% Glucose   Autoclave
10% (w/v) Glucose
 
BioReagents and Chemicals
Ammonium Sulfate
HEPES
IPTG
Glucose
Liquid nitrogen
DTT
Glycine
EDTA
Sodium Chloride
PIPES
Yeast Extract
Sodium Acetate
Nickel (2+)-Nitrilotriacetic Acid Affinity Resin
Glycerol
Potassium Hydroxide
Guanidine
Tryptone
Sodium Hydroxide
 
Protocol Hints
1. The optimal IPTG concentration must be empirically determined. A good concentration to start with is 0.3 mM.

2. Pellet can be stored at this point frozen. After thawing the pellet, redissolve it in 1 ml Guanidine solution, pH 7.9.

3. You should obtain approximately 5 mg purified protein from 100 ml E. coli culture.

 
Citation and/or Web Resources
1. Ge H, Zuo P, Manley JL. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell 1991 Jul 26;66(2):373-82.