Tagged protein purification is a robust protein purification method. It relies on the interaction between a fusion protein (Tag + Target protein) and its specific ligand immobilized on the resin. This method is usually employed to obtain highly pure protein solutions and to concentrate proteins present at low concentrations in complex mixtures.

Please note that tagged protein purification cannot be ordered as a standalone service but can be part of our protein expression services.

What are the Advantages of Tagged
Protein Purification?

  1. Fast Purification: Affinity tags’ high selectivity to target the protein of interest from a complex protein mixture allows one-step purification. In other words, expressed proteins can directly be passed through the chromatography column altogether with crude cell lysate, media, and present contaminants.
  2. Specific Purification: Tagging recombinant proteins allows specific separation of the protein of interest from all the present molecules in the mixture. This feature is acquired thanks to the high affinity between the tag and its ligand.
  3. High Resolution: Tag-affinity protein purification is well-known for its high resolution. It is translated by an effective separation of the protein of interest, easily detected on the elution graph.

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Affinity Purification Techniques

Tag-affinity purification is one of the most common protein purification techniques. The former is based on the high affinity created between the fusion protein and its specific ligand. This method grants important purity levels. For instance, over 90% is easily achievable with this technique. Much higher purity percentages can even be achieved when other purification techniques are combined.

Affinity purification tags simplify the downstream processing into a single-step purification and increases yields through the specific interactions between ligands and fusion proteins. There are several types of protein tags. Yet, not all tags are suitable for all protein types and all downstream applications.

Affinity Tags

These tag types are small in size and rarely alter the protein’s structure and function. They allow specific protein separation from a complex protein mixture.  To mention some examples:

  • Poly-His-Tag: His-tag is used with a wide range of protein expression systems. It involves a one-step IMAC chromatography purification. As a result of the non-specific adsorption of metalloproteins and other non-specific molecules, only medium purity levels are attained with this tag.
  • Twin-Strep-Tag: Compared to poly-his-tag, twin-strep-tag offer much higher purity levels without the need for further purification steps. The gentle chromatography conditions and the physiological buffer make this tagging method suitable for heterodimeric protein purification and functional studies.

Solubility Tags

In addition to their purification properties, solubility tags protect tagged proteins from proteolysis and enhance their solubility. However, their larger size adds more complexity to the downstream process. It is therefore important to cleave the tag at the end of the purification and eliminate the risk of altering the natural conformation of the protein of interest.

  • GST-Tag: This tag is adapted to complex proteins difficult to obtain in soluble forms. It protects them from proteolytic degradation and can be employed in various expression systems.
  • MBP-Tag: MBP-tag is usually used to increase the expression levels of the protein of interest. It also guarantees the stability of proteins and protects them from proteolytic degradation. However, the cleavage of the MBP-tag is not as effective as for GST-tag.


They are short amino acid sequences that rarely interfere with the protein conformation and activity. Epitopes serve for antibody purification and protein detection.

  • FLAG: It is generally considered a universal tag and can capture the FC region of immunoglobulins.
  • c-Myc: This tag is reliable for various applications such as Immunoprecipitation (IP), Western Blotting (WB), Immunofluorescence (IF)…
  • Protein A: Protein A grants highly specific affinity to the antibody’s FC region. Moreover, the former target a variety of species from humans to mice, donkeys, rabbits…

Protein Purification By Affinity Chromatography

What Is Affinity Purification?

Affinity chromatography is a biochemical purification technique. It relies on reversible interactions between a protein and his ligand. The goal behind this technique is to isolate proteins of interest specifically from all other molecules allowing their purification from a heterogenous solution.

This type of chromatography is generally composed of a mobile phase and a stationary phase.

  • Stationary phase: Protein-specific ligands immobilized to the chromatography’s matrix. Examples: Antibodies, antigens, ions, molecules…
  • Mobile phase: Crude cell lysate including the protein of interest. Example: An antibody solution with cell fragments and other proteins.

Principle Of Affinity Chromatography:

3 major steps compose affinity chromatography process:

  • Binding: The crude cell lysate is poured into the column. The targeted protein is bound to the ligands.
  • Purification: Eliminating the unbound elements and the non-specific bindings by several buffer washes.
  • Elution: Elution of a pure protein by a specific buffer to break off the bonds created with ligands. The protein is released.

Successful protein purification by affinity chromatography relies highly on the tags and ligands used. The latter are responsible for the affinity to the proteins. Moreover, they provide specific interactions and stability to the process.

Here are most of the common affinity purification techniques:

  • Tag-Assisted Chromatography:

Protein tags can vary from a short peptide sequence to a protein. They are synthesized along with the protein of interest in a way to be fused during the expression. Protein tags are mainly used to promote the protein purification process. Tags constitute an attractive approach as they simplify the downstream process into a single-step purification process. They can also serve in several other applications like protein characterization, and interaction studies. And bring in further advantages to the protein such as solubility and stability.

  • Immobilized Metal Affinity Chromatography (IMAC): A widely used method for antibody and recombinant protein purification. As indicated by its name, this technique uses immobilized metal ions as ligands. IMAC offers the advantage to purify inclusion bodies under denaturing conditions. The proteins are then refolded using specific reagents. This technique facilitates the purification of his-tagged recombinant proteins.
Tag Type Tag Name Origin Size (aa) Interfering Localization Matrix
Affinity Poly-Histidine Synthetic ~6 Rarely N-terminal
C- terminal
Immobilized metal ions: Ni2+, Co2+, Cu2+, Zn2+.
Affinity Strep II-tag Bacteria 8 No N-terminal
C- terminal
Affinity Poly-Arginine Synthetic 5 Yes C- terminal Cation exchange resin.
Affinity Calmodulin Binding Protein- CBP Skeletal muscle myosin light chain kinase 26 No N-terminal
C- terminal
Immobilized calmodulin.
Solubility Glutathione-S-Transferase-GST S.japonicum 211 Yes N-terminal Glutathione resin.
Solubility Maltose-Binding Protein-MBP E.coli 396 Yes N-terminal
C- terminal
Immobilized amylose or maltose resins.
Solubility Small Ubiquitin-Like-Modifier- SUMO H.sapiens ~100 No N-terminal Catalytic core of Ulp1.
Solubility N-utilization substance-NusA E.coli 495 Yes N-terminal
C- terminal
Epitope FLAg tag Peptide- FLAG Synthetic 8 Yes N-terminal
C- terminal
Anti-FLAG antibody.
Epitope c-MYC H.sapiens 10 N/A N-terminal
C- terminal
9E10 antibody coupled with agarose.
Epitope EPEA / C-tag Synthetic 4 Minimal C-terminal Erbin PDZ domain and immobilized on the agarose beads.
Epitope Hemagglutinin (HA) Human influenza HA molecule 31 N/A N-terminal
C- terminal
Monoclonal antibodies.
Fluorescence Green Fluorescent Protein (GFP) Aequorea victoria 220 Yes N-terminal
C- terminal
Fluorescence S-tag RNase A 15 No N-terminal
C- terminal
S-fragment of RNase A.
Fluorescence mCherry DsRed of Discosoma 256 N/A C- terminal Affinity purified by Protein A.
  • Immuno-Affinity Chromatography (IAC):
  • Thanks to immobilized antibodies, IAC can achieve highly specific purification. This technique is usually employed to identify and purify antigens and viral proteins. On the other hand, IAC can also serve for antibody purification, by replacing immobilized antibodies with immobilized antigens or proteins. In this context, protein A/G constitutes a popular choice for antibody purification.

  • Protein A/G Affinity Chromatography: Antibody purification constitutes an efficient method and provides one-step purification. It is a popular technique for IgG purification as protein A/G has a high affinity for the antibody’s Fc region. Therefore, protein A/G chromatography can deliver almost homogenous antibodies. The former is also validated to use with GMP processes. Moreover, Protein A/G affinity chromatography targets a variety of species from humans to mice, donkeys, rabbits, and more.
  • Lectin Affinity Chromatography: Lectin affinity chromatography constitutes an inexpensive and stable technique. It is generally used for glycoprotein purification. Since membrane proteins are composed of carbohydrate compartments, these proteins have a high affinity to the immobilized lectin.

To summarize, tagged protein purification offers affinity, specificity, and a fast purification process.