The strong, specific, and well-characterized interaction between the Fc part of immunoglobulin G (IgG) and Staphy-lococcus aureus protein A (SpA) was the first biointeraction exploited for the creation of an affinity fusion system allowing affinity chromatography purification of expressed recombinant gene products (1). To date, hundreds of proteins have been produced as SpA fusions in a wide variety of host cells (2-5). During the past decade a large number of additional affinity fusion systems have been presented (for reviews, see 2,6-9). Affinity fusions are primarily used to simplify recovery of a fused target gene product but are also widely employed for detection and immobilization purposes (10). Most affinity tags are easy to use as detection moieties to monitor the production or distribution of a produced fusion protein, and certain affinity tags are particularly suited to obtain efficient and directed immobilization of a target protein.

This article describes the use of two affinity tags, the IgG-binding SpA domains and the serum albumin-binding region of streptococcal protein G (SpG), which both carry a number of features that make them highly suited as fusion partners in different applications. The wide spectrum of applications for affinity fusions in biotechnology is illustrated by selected examples in which SpA or SpG fusion proteins have been used to (1) improve the stability to proteolysis of produced recombinant proteins, (2) allow integrated downstream processing schemes by careful genetic design, (3) improve in vitro renaturation schemes for expressed gene products, (4) enable affinity-assisted folding in vivo of target proteins, (5) prolong the in vivo half-life of therapeutic proteins, (6) facilitate subunit vaccine development and functional cDNA analysis, and (7) select novel receptor variants with new specificities by the use of phage display technology.

50 affinity fusions, gene expression COMMONLY USED AFFINITY TAGS

A large number of different affinity fusion systems have been described involving fusion partners that range in size from one amino acid to large multisubunit proteins capable of selective interaction with a ligand immobilized onto a chromatography matrix. Different types of interactions have been utilized, such as enzymes-substrates, bacterial receptors-serum proteins, polyhistidines-metal ions, and antibodies-antigens (2). The degree of specificity in the interaction and conditions for the affinity purification differ from system to system. The environment tolerable by the target protein is an important factor for deciding which affinity fusion system to choose. Also, other factors, including costs for the affinity matrix and buffers and the possibilities for column sanitation and reuse, are important to consider. Numerous gene fusion systems for affinity purification have been described in the literature, all with different characteristics (2,6-9). Some of the most commonly used systems are listed in Table 1, and some relevant references are given for each system. These different affinity fusion systems, with their advantages and disadvantages, were described in a recent review (10). The polyhistidyl tags, which suffer from a somewhat lower specificity, have the advantage of allowing affinity purification under denaturing conditions. The in vivo biotinylated "Bio-tag" is particularly suited for immobilization purposes because of the strong interaction (association constant; KA ^ 1015 M_1) between biotin and streptavidin, but is inconvenient to use for affinity purification purposes because efficient elution is difficult to accomplish. One of the most extensively used affinity tags is the glutathione S-transferase (GST) from the parasitic helminth Shistosoma japonicum (17). GST fusion proteins can be purified using glutathione as immobilized ligand, also following renaturation from inclusion bodies after solubilization with 6 M guanidinium hydrochloride (18). A possible complication associated with the GST fusion system is the use of reduced glutathione (a reducing agent) for elution, which can affect target proteins containing disulfides (10).

Although a multitude of systems have been described, no single affinity fusion strategy is ideal for all expression or purification situations. For example, if secretion of the product into the periplasm or culture medium is desired, fusion to affinity tails derived from normally intracellular proteins (e.g., the j-galactosidase system) is not applicable, and if the purification must be performed under denatur-ating conditions, protein ligands such as monoclonal antibodies (mAbs; e.g., anti-FLAG M1 and M2 mAbs) are unsuitable. Therefore, to obtain general expression vectors for affinity gene fusion strategies applicable in several situations, a combination of affinity fusion domains could be introduced into a single fusion partner. Such composite fusion partners, consisting of several independent affinity domains, could potentially be used in different detection, purification, and immobilization situations, employing the affinity function that is most suitable for the situation. However, the included affinity domains should be carefully chosen not to interfere functionally with each other, and each moiety should be able to withstand the purification conditions dictated by the affinity domain requiring the harshest affinity chromatography conditions. A further advantage using combined affinity tags is that different methods for the detection and immobilization of the fusion protein also can be envisioned.

Recently, a new affinity fusion partner was described in which this strategy was utilized (26). An affinity-tag combination was designed consisting of the in vivo biotinylated peptide (Bio), a hexahistidyl peptide (His6), and the SpG-derived albumin-binding protein (ABP) that could allow for flexible use in different detection, purification, and immobilization situations (26). Fusions to the Bio-His6-ABP affinity tag could be used for easy detection using commercial streptavidin conjugates, for purification under both native and denaturating conditions, and for directed immobilization purposes with an option to choose among three strong affinity interactions. In addition, the ABP part of the affinity tag is highly soluble and effectively refolds after denaturation, which can be an advantage during refolding of the target protein fused to the Bio-His6-ABP affinity tag. Such composite affinity tags have the potential of becoming widely used in the future because they offer flexibility.

This article does not focus on the description of different characteristics of various affinity-tag systems, as up-to-date reviews are available (4,9,10), but instead describes various application areas in which affinity fusions to the

Table 1. Commonly Used Affinity Fusion Systems

Fusion partner





Protein A

31 kDa


Pharmacia Biotech


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