Overview of affinity tags for protein purification

Abstract

Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future.

Keywords: affinity chromatography; affinity tag; epitope tag; protein detection; protein purification.

Figure 9.9.1

Flow chart describing the general scheme for choosing an affinity tag for protein purification if a larger amount (10–100 mg) of highly pure untagged protein is required. Experimental methods requiring this high quantity of pure, native protein include, but are not limited to, protein crystallography, NMR spectroscopy, isothermal titration calorimetry, and surface plasmon resonance. Solid lines indicate forward progression in purification optimization, dashed lines indicate steps backwards, and thick boxes indicate the final desired product, a protein of high purity and quality.

Figure 9.9.2

Flow chart describing the general scheme for selecting an affinity tag for protein purification if a mid-range amount (0.1–10 mg) of high purity tagged and/or untagged protein is needed. An example of an experimental method requiring this quantity of tagged protein is surface plasmon resonance (SPR), where a biotin moiety could be sued to attach a protein ligand to a streptavidin-coated SPR chip. Other possible applications are in GST and Ni²⁺ precipitation assays, and mass spectrometry. Certain variants of the intien-CBD domain are useful for methods requiring a completely native protein. Finally, the His-tag and GST-tag systems are useful because both tagged and untagged proteins can be easily prepared from the same cellular lysate, and there exist many secondary reagents specifically for use with these two systems. Thick lines indicate the desired result has been reached.

Figure 9.9.3

Flow chart describing the general scheme for selecting an affinity tag for purification of a small amount (<0.1 mg) of moderate-to-high purity tagged protein. Such a protein might be used in co-immunoprecipitation, Western blotting, far-Western blotting, ELISA, or gel overlay assays. Since only small amounts of protein are required, it is not usually necessary to optimize protein expression, leaving the choice of affinity tag to be based on the final use for the fusion tag—i.e., as a reporter or for general detection. Examples of epitope tags include the HA-tag, FLAG-tag, T7-tag, Myc-tag, and others.