When expressing and purifying large quantities of soluble protein, expression difficulties often include poor yield and the formation of insoluble aggregates. Gene fusion technologies can overcome these obstacles and simplify purification and improve solubility. This article discusses the most popular fusion tags and the enzymes used to remove them, with special reference to recently introduced technologies.
Affinity tags are the most commonly used tag for aiding in protein purification. They can be defined as exogenous amino acid (aa) sequences that bind with high affinity to a chemical ligand or an antibody. Most affinity tags are short peptide sequences that either bind to a ligand linked to a solid support (like the His tag) or contain an epitope recognized by immobilized antibodies (like the FLAG or Myc tags). The high affinity of these tags for their ligands and the availability of well developed immobilized supports for capturing the fusion proteins allow the protein of interest to be purified to a very high degree. Because of their small size, these affinity tags can be added at either end of the protein or in a region that is exposed to the surface. However, these tags generally do not increase the expression of the fusion proteins or enhance their solubility, and therefore are of little use in purifying hard-to-express proteins.
His-tags are the most widely used affinity tags. The purification of his-tagged proteins is based on the use of a chelated metal ion as an affinity ligand; one commonly used ion is the immobilized nickel-nitrilotriacetic acid chelate [Ni–NTA], which is bound by the imidazole side chain of histidine. Similarly, Streptag II, which consists of a streptavidin-recognizing octapeptide (WSHPQFEK), can be purified by affinity using a matrix with a modified streptavidin and eluted with a biotin analog. Other commonly used affinity tags like FLAG, Myc, and HA can be purified by binding to respective antibodies immobilized on chromatographic supports.
Because it is desirable to remove most tags at the end of the purification process, considerable advances have been made in design of affinity tags so that they can be cleaved without leaving any residues behind and also to simplify the entire process of purification and cleavage. One such system is the "Profinity eXact" fusion-tag system (Bio-Rad, Hercules, CA), which uses an immobilized subtilisin protease to carry out affinity binding and tag cleavage. The protease is not only involved with the binding and recognition of the tag, but upon application of the elution buffer, it also serves to precisely cleave the tag from the fusion protein directly after the cleavage recognition sequence. This delivers a native, tag-free protein in a single step. Another system for simple purification of proteins is based on elastin-like polypeptides (ELP) and intein. ELP consist of several repeats of a peptide motif that undergo a reversible transition from soluble to insoluble upon temperature upshift. The fusion protein is purified by temperature-induced aggregation and separation by centrifugation, and intein is used for tag removal.3 No affinity columns are needed for initial purification.