Abstract
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.
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Recombinant proteins show large variability in terms of their expression, solubility, stability, and functionality, making
them difficult targets for large-scale analyses and production. Advances in recombinant protein expression include the development
of better expression systems and host strains, improving mRNA stability, host-specific codon optimization, the use of secretory
pathways, post-translational modification, co-expression with chaperones, and decreasing the amount of proteolytic degradation.
However, no other technology has been as effective in improving the expression, solubility, and production of biologically
active proteins as the addition of fusion tags, especially for difficult-to-express proteins.
Genetically engineered fusion tags allow the purification of virtually any protein without any prior knowledge of its biochemical
properties.1–2 They can improve the variable yield and poor solubility of many recombinant proteins. Proper design and judicious use of
the right fusion tag can enhance the solubility and promote proper folding of the protein of interest, leading to recovery
of more functional protein. On the other hand, adding fusion tags has been reported to result in changes in protein conformation,
poor yields, loss or alteration of biological activity, and toxicity of the target protein. For this reason, it is desirable
to remove the tag from the target protein after expression. When designing a fusion tag, therefore, careful consideration
must be given to how the tag will be removed to produce native proteins without any extraneous sequences. Cleavage of the
tags and the proteases that are used to cleave the tag are discussed in the next section.
Table 1. Commonly used fusion tags for purification and enhancing the solubility of proteins
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Many fusion tags are available for the expression and purification of proteins (Table 1). These tags can be broadly classified
into two categories: affinity tags that aid in purification but do not enhance the solubility of the proteins substantially, and solubility-enhancing tags that specifically enhance the solubility and recovery of functional proteins.
Affinity Tags
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.
