Affinity tag protein purification is a widely used method for isolating recombinant proteins from complex mixtures, such as cell lysates, by using specific affinity tags that facilitate the purification process. Affinity tags are short peptide sequences or proteins genetically fused to the target protein, allowing efficient capture and elution using a corresponding ligand or binding partner.
Common Affinity Tags
His-Tag (Polyhistidine Tag):
Structure: A sequence of 6–10 histidine residues.
Binding Partner: Metal ions (Ni²⁺, Co²⁺) immobilized on a matrix (Ni-NTA, Co-NTA).
Elution: Imidazole, which competes with histidine for metal binding, or by lowering the pH.
Advantages: Simple, efficient, works under native and denaturing conditions.
Applications: Often used for bacterial, yeast, and mammalian protein expression.
GST-Tag (Glutathione-S-Transferase Tag):
Structure: A 26 kDa protein that binds to glutathione.
Binding Partner: Glutathione immobilized on resin.
Elution: Free glutathione.
Advantages: Can improve protein solubility and stability.
Applications: Ideal for purifying proteins expressed in bacterial systems.
FLAG-Tag:
Structure: A short, hydrophilic sequence (e.g., DYKDDDDK).
Binding Partner: Anti-FLAG antibody or FLAG peptide.
Elution: Competitive elution using FLAG peptide or by altering pH.
Advantages: Small size, minimal impact on protein folding or function.
Applications: Suitable for various expression systems, including mammalian cells.
Strep-Tag:
Structure: A short peptide sequence that binds to streptavidin or Strep-Tactin.
Binding Partner: Strep-Tactin or streptavidin resin.
Elution: Biotin or desthiobiotin.
Advantages: High specificity, mild elution conditions.
Applications: Used in sensitive applications such as protein complex isolation.
MBP-Tag (Maltose-Binding Protein):
Structure: A 42 kDa protein that binds maltose.
Binding Partner: Maltose immobilized on amylose resin.
Elution: Free maltose.
Advantages: Can enhance solubility and folding of target proteins.
Applications: Used for proteins that are difficult to express or tend to aggregate.
HA-Tag (Hemagglutinin Tag):
Structure: A short peptide sequence derived from the influenza virus hemagglutinin protein (e.g., YPYDVPDYA).
Binding Partner: Anti-HA antibody or resin.
Elution: Competitive elution using HA peptide or mild buffer conditions.
Advantages: Small and minimally affects protein structure or function.
Applications: Commonly used in mammalian cell systems for immunoprecipitation or Western blotting.
General Steps in Affinity Tag Protein Purification
Construct Design and Protein Expression:
The gene encoding the target protein is fused with the gene for an affinity tag, either at the N-terminus or C-terminus of the protein.
The recombinant tagged protein is expressed in a suitable expression system (e.g., E. coli, yeast, or mammalian cells).
Cell Lysis:
Cells expressing the tagged protein are lysed to release the target protein. The lysis method should be mild enough to preserve protein structure and activity.
Common lysis methods include sonication, detergents, or enzymatic lysis (e.g., lysozyme in bacterial cells).
Binding to Affinity Resin:
The lysate is incubated with an affinity resin (e.g., Ni-NTA for His-tag, glutathione resin for GST-tag) that specifically binds to the affinity tag.
The tagged protein, along with any interacting partners (if applicable), binds to the resin, while unbound proteins are washed away.
Washing:
The column is washed with binding buffer to remove non-specifically bound proteins and contaminants.
Wash buffers may contain low concentrations of eluting agents (e.g., imidazole for His-tagged proteins) to reduce background binding.
Elution:
The tagged protein is eluted from the column by altering the buffer conditions. Elution can be achieved by:
Competitive binding (e.g., imidazole for His-tag, free glutathione for GST-tag).
Changes in pH or ionic strength.
Adding specific eluting peptides (e.g., FLAG peptide for FLAG-tag).
Tag Removal (Optional):
If the tag affects the protein’s function or structure, it can be removed using a protease (e.g., TEV or thrombin) if a protease cleavage site is included between the tag and the protein.
After cleavage, the tag and protease can be removed by a second round of affinity purification or size exclusion chromatography.
Protein Analysis:
The eluted protein is analyzed for purity using SDS-PAGE, Western blotting, or other biochemical methods.
The protein may be further characterized by enzymatic assays, structural analysis, or mass spectrometry.
Dialysis and Storage:
The purified protein is dialyzed to remove elution reagents and adjust buffer conditions.
The protein is then concentrated and stored at 4°C (short term) or -80°C (long term), often in the presence of stabilizing agents such as glycerol.
Example Protocol for His-Tag Protein Purification Using Ni-NTA Resin
Materials
Ni-NTA resin
Lysis buffer: 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.4
Wash buffer: 50 mM sodium phosphate, 300 mM NaCl, 20–30 mM imidazole, pH 7.4
Elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 250–500 mM imidazole, pH 7.4
Procedure:
- Cell Lysis:
Harvest cells expressing the His-tagged protein and resuspend them in lysis buffer.
Lyse cells using sonication or detergent-based lysis and centrifuge to remove debris.
- Equilibrate Resin:
Wash the Ni-NTA resin with binding buffer (lysis buffer) to equilibrate it.
- Binding:
Add the cleared lysate to the equilibrated Ni-NTA resin and incubate at 4°C for 1–2 hours to allow the His-tagged protein to bind.
- Washing:
Wash the resin with 10 column volumes (CVs) of wash buffer to remove unbound proteins.
- Elution:
Elute the bound His-tagged protein by applying elution buffer with 250–500 mM imidazole. Collect the eluate in small fractions.
- Analysis:
Analyze the eluted protein by SDS-PAGE to assess purity and concentration.
- Optional Tag Removal:
If the tag needs to be removed, add protease (e.g., TEV protease) to cleave the tag and re-purify the protein by size exclusion chromatography or affinity chromatography to remove the tag.
- Storage:
Dialyze the purified protein into a suitable buffer and store at -80°C for long-term use.
Applications of Affinity Tag Protein Purification
- Recombinant Protein Production: Allows for easy and efficient purification of proteins for structural biology, functional assays, or therapeutic development.
- Protein-Protein Interaction Studies: Purified proteins can be used in interaction assays, including co-immunoprecipitation, pull-down assays, and affinity purification-mass spectrometry (AP-MS).
- Structural Studies: High-purity protein is essential for crystallization, NMR, and cryo-EM studies to determine protein structure.
- Functional Studies: Enzymatic or biochemical assays often require high-purity proteins, and affinity tags enable quick and reproducible purification.
Affinity tag-based purification is an indispensable tool in molecular biology, enabling researchers to rapidly and efficiently isolate target proteins for a wide range of applications.