Polyhistidine binds to a variety of transition metals and transition metal chelates, so proteins with exposed His-6 bind to cured Ni2+ resins, and target proteins can be recovered by rinsing with an appropriate buffer to remove other proteins and then eluting with a soluble competitive chelator. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.

Operation method

Experiments on the purification of proteins with histidine tags by solidified Ni2+ absorption chromatography

Materials and Instruments

E. coli cells expressing proteins with polyhistidine tags
Binding buffer Imidazole elution buffer TritonX-100 wash buffer SDS-polyacrylamide gel
Sorvall SS-34 Turntable or equivalent Chromatography column cured Ni2+ Chromatography Resin Shake table

Move

makings

Buffers and solutions

Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.

Binding buffer (pH 7.8)
20 mmol/L sodium phosphate
500 mmol/L NaCl

Imidazole elution buffer (pH 6.0)
20 mmol/L sodium phosphate
500 mmol/L NaCl
In the washing buffer (pH 6.0), 3mol/L imidazole was added to form 10 mmol/L, 50 mmol/L, 100 mmol/L and 150 mmol/L imidazole elution buffer.

TritonX-100 (10%V/V)

Wash buffer (pH 6.0)
20 mmol/L sodium phosphate
500 mmol/L NaCl

Enzyme and buffer
DNase (1 mg/ml)
Lysozyme
RNase (1 mg/ml)

Gel

SDS-polyacrylamide gel (10%)
Refer to Appendix 8 for preparation of SDS-polyacrylamide gels for protein isolation.

Centrifuge and rotor head

Sorvall SS-34 rotor head or equivalent

Special equipment

Chromatography columns (glass or polypropylene)

Cured ^Ni2+ chromatography resin (e.g., ProBond, Invitrogen; HisTrap, Pharmacia; His.Bind Resin, Novagen; ^{NTA-Ni2+-agarose}, Qiagen)
The most commonly used metal chelating affinity carrier today is hypromellose acetate (NTA) ^{-Ni2+-agarose}, which can be purchased from the above companies, as well as other carriers of different properties. A unique ^Co2+ chelate (Talon) is said to have a higher affinity and selectivity for His-6, but it is not easy to regenerate in the laboratory. It is therefore more expensive.

Shaker

Carriers and Bacterial Strains
E. coli cells expressing proteins with polyhistidine tags (generated by protocols 1, 2, 3, or 4).

Methods

Preparation of ^Ni2+ affinity columns

1. Mix the cured ^Ni2+ resin by gently inverting it and load 2 ml of it onto the column and allow the resin to settle naturally.

2. Rinse the resin with 3 times the column volume of sterile water.
The volume of resin after settling is 1 column volume.

3. Equilibrate resin with 3x column volume of Binding Buffer (pH 7.8) and allow to stand until step 9.

Preparation of Cell Extracts

4. Suspend 4 ml of Binding Buffer (pH 7.8) per 100 ml of cell culture.

5. Add lysozyme to a final concentration of 1 mg/ml and leave on ice for 30 min.
In some protocols, cells are lysed by sonication or French pressure prior to affinity purification of histidine-tagged proteins. The method using lysozyme is more reproducible and does not cause catastrophic cell lysis, which releases outer membrane proteins and other molecules that can cause aggregation of histidine-tagged proteins.
Note: Protease inhibitors can be added, but not EDTA or other chelating agents, as they bring ^Ni2+ off the resin. This destroys the affinity of the resin for histidine.

6. Incubate the mixture on a shaker at 4°C for 10 min.

7. Add Triton X-100, DNase and RNase at final concentrations of 1%, 5ug/ml and 5ug/ml, respectively, and incubate for 10 min at 4°C on a shaker.

8. Remove insoluble cellular debris by centrifugation at 3000 g (5000 r/min Sorvall SS-34 turning head) for 30 min at 4°C. The supernatant (cell lysate) was transferred to a new tube.
To prevent clogging of the resin during purification, the supernatant should be filtered through a 0.45 membrane.

Purification of histidine-tagged proteins

9. Drain the binding buffer from the resin to the top of the column.

10. Cell lysate supernatant obtained in step 8 is loaded onto the column and the flow rate is adjusted to 10 columns per hour.
1 ml of ^Ni2+ affinity resin binds approximately 8-12 mg of protein. The yield of polyhistidine-tagged proteins in E. coli varies depending on the target protein, but is generally in the range of per 100 ml of culture protein .

11. Wash the column with 6 column volumes of binding buffer (pH 7.8).

12. Wash the column with 4 column volumes of Wash Buffer (pH 6.0) until the flow-through solution _A280 is <0.01.

13. Elute bound proteins with 6 column volumes of 10 mmol/L Imidazole Elution Buffer, 1 ml of each to be collected in stepwise fashion and monitor A280.

14. Repeat step 13 with higher concentrations of imidazole (50 mmol/L, 100 mmol/L, and 150 mmol/L).
Bound proteins can also be eluted with 10-100 mmol/L continuous gradient imidazole elution buffer, with most histidine-tagged proteins eluting between 50 and 100 mmol/L. If imidazole is not used, it can also be used to elute A280. If imidazole is not used, a pH-descending buffer can also be used (see Alternative: Elution of Polyhistidine-Tagged Proteins from Metal Affinity Columns with pH-Descending Buffer).

15. Analyze the distribution of polyhistidine-tagged proteins by 10% SDS polyacrylamide gel electrophoresis using a 20ul aliquot of protein.

Common Problems

This experiment was derived from the next volume of Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.

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