Protein purification based on fusing peptide affinity tag to recombinant protein is widely preferred due to its ease of use. Immobilized Metal Ion Affinity Chromatography (IMAC) was based on the principle of binding of specific amino acid side chains of a protein to metal ions immobilized on a matrix. Histidine amino acid has high affinity to metal ions, six histidine residues are fused to N-terminal or C-terminal end of a protein during cloning, and the protein obtained could be ~95% pure after passing through metal ion based resin. Protein purification based on metal affinity chromatography is used with all the expression systems including E.coli, Yeast, Insect cell and mammalian cell expression systems.
Types of His Tags
- Small peptide tags usually have less effect on the protein of interest, so a 6X Histidine tag (HHHHHH) is preferred. However, increasing the number of histidines results in increased affinity. It can be used when purifying proteins under both denaturing and non-denaturing conditions.
- Histidine-Glutamine tag is 6-10 amino acids in length with alternative Histidine and glutamine residues (HQHQHQ). Its function is similar to 6X His tag except for the elution of HQ tagged proteins is done at 50mM imidazole concentrations. It can also be stable at both denaturing and non-denaturing buffer conditions.
- Histidine- Asparagine tag is a small peptide tag with alternative Histidine and Asparagine residues (HNHNHNHNHNHN). It has a high metal binding affinity compared to 6x His-tag.
- HAT tag: This is a 19 amino acid long natural Histidine affinity tag derived from chicken L-Lactate dehydrogenase A chain (P00340 UniProt ID) with sequence —KDHLIHNVHKEEHAHAHNK. HAT Tag is eluted with low pH buffer or buffer with 150mM imidazole.
Metals Ions
Nickel, Zinc, Cobalt and Copper are the transition metals that are chelated to matrices, such as Iminodiacetic acid (IDA), Nitrilotriacetic acid (NTA), Carboxymethylaspartate (CMA) resins. Ni-IDA, Ni-NTA, Co-CMA are used widely. NTA has strong affinity compared to IDA and CMA. NTA resins are stable and bind the metal tightly even after multiple regeneration steps.
Eluent
Proteins can be eluted from the resin by decreasing the elution buffer pH to 4.00- 6.00. Low pH conditions trigger the decrease of affinity between histidines and metals resulting in a weak bond between protein and metal. Low pH is not suitable for some proteins resulting inactive protein or denaturaed protein as a final product. Protein can also be eluted by addition of free imidazole at a concentration of 250 mM to elution buffer. This is compatible for all proteins.
Non-specific protein binding is one of the major problems faced during protein purification in addition to weak binding of proteins.
Other points to consider
- Addition of 10-20 mM imidazole in wash buffers to remove non-specific binding proteins.
- Addition of Non-ionic detergents like 0.1% Triton X-100 or 0.1%Tween-20 in wash buffers to avoid non-specific binding.
- Addition of 5-10% glycerol for protein stability and non-specific binding.
- Addition of DTT or BME at 1mM concentration for purifying proteins that can form disulphide bonds with other proteins.
- Avoid using metal chelators like EDTA in all purification buffers to avoid metal chelation from the matrix.
- Addition of denaturants like Guanidine HCl or Urea at low concentrations (1-2M) helps in purifying proteins with affinity tags that are buried inside the protein during protein folding.
References
- Godat B1,"Engel L,"Betz NA,"Johnson TM. Methods for the purificationof HQ-tagged proteins; Methods Mol Biol."2008;421:151-68.
- Joshua A. Bornhorst and Joseph J. Falke. Purification of Proteins Using Polyhistidine Affinity Tag; Methods Enzymol. 2000 ; 326: 245–254.
