Z

an affinity tag and subsequently used for specific cleavage of a target protein (108). This enabled removal of the affinity-tagged protease and the cleaved affinity fusion partner by simple affinity chromatography steps. It was in addition possible to add any desired amount of protease to ensure efficient cleavage because the protease could be easily removed after cleavage (108). Recently, a different affinity-tagged protease, consisting of coxsackievirus 3C (3Cpro) fused to the serum albumin-binding ABP at the N-terminus and His6 at the C-terminus, was produced in E. coli and used for the production of a truncated Taq DNA polymerase (DTaq) (102). The heat-stable polymerase was produced as an ABP-D Taq fusion protein having a 3Cpro cleavage site introduced between the two protein moieties. After affinity purification of ABP-D Taq, the fusion was efficiently cleaved using the affinity-tagged protease ABP-3Cpro, which allowed for subsequent recovery of nonfused, fully active D Taq via passage of the cleavage mixture over an HSA-column (102). Note that the affinity-tagged protease, ABP-3Cpro, is removed together with eventual remains of unprocessed ABP-D Taq fusion protein.

The use of affinity fusions for recombinant production of therapeutic target gene products has probably been hampered by the difficulties with efficiently cleaving off the affinity tag and thereafter removing not only the tag but also the used protease. The increasing availability of affinity-tagged, highly specific proteases for cleavage of fusion proteins to enable straightforward methods for recovery of unfused authentic gene products (102,108) will probably influence researchers to further increase their use of fusion strategies for production of target proteins.

Increased In Vivo Half-Lives for Therapeutic Gene Products

Gene fusion technology might also prove to be useful in prolonging the in vivo half-life of pharmaceutical proteins by administering the therapeutic protein as fused to a protein with extended half-life. The principle was demonstrated to be efficacious by Capon and coworkers (109), who showed that by fusing a soluble portion of CD4 (the target receptor for the human immunodeficiency virus particle) to the Fc part of IgG, the serum half-life of CD4 in rabbits increased 200-fold. Nygren and coworkers (110) described an alternative strategy where in vivo stabilization was achieved by fusion of the unstable CD4 protein to BB, leading to the formation of complexes between the fusion protein and serum albumin. The serum half-lives of different fusion proteins containing the albumin-binding receptor were tested in mice. In these studies, the half-life of the CD4 fusion in vivo was shown to be comparable to the CD4-IgG molecule described earlier. Interestingly, the albumin-binding receptor in macaque monkeys was found to have a serum half-life similar to that of serum albumin itself (110). Recently, the same strategy was employed to increase the serum half-life of human soluble complement receptor type 1 (sCR1) in rats, by fusion to different serum albumin-binding fragments of SpG (81). These examples suggest that gene fusions to BB, ABP, or the minimal binding motif ABD of SpG might constitute useful strategies to improve the in vivo stability of pharmaceutical proteins. It may also be possible to combine these approaches with new methods for drug targeting or slow drug release.

Protein A Fusions in Immunodetection Techniques

Bifunctional SpA fusion proteins have found extensive use for various analytical purposes, for example, in enzyme im-munoassays. Protein A fusion proteins can after expression be efficiently purified using the IgG-binding activity, and in most assays the protein A moiety binds antibodies to be quantified and the activity of the fused gene product is monitored. Thus, the fusion protein can replace more traditional reagents, such as secondary antibodies conjugated to an enzyme. The concept of genetically engineered conjugates in immunoassays was recently reviewed (111). The fusion between catechol dioxygenase and protein A produced in E. coli was used in an enzyme immunoassay to quantify antibodies (112). The principle of a bioluminescent immunoassay using the fusion between protein A and bacterial luciferase has also been demonstrated (113), and recently a bifunctional fusion between domain D of SpA and luciferase from sea-firefly was used in a sandwich enzyme-linked immunosorbent assay (ELISA) (72). Furthermore, an SpA-streptokinase fusion was shown to be equally useful as a commercial SpA-horseradish peroxi-dase conjugate for use in immunoassays (60). A fusion between SpA and LacG, 6-phospho-ö-galactosidase from S. aureus, was shown to be able to replace secondary antibodies in an ELISA (114). In a similar approach, an SpA-alkaline phosphatase fusion was used in ELISA (115) and a fusion between maltose-binding protein and protein A was used for a solid-phase immunoassay, in which the fusion protein was immobilized to determine IgG concentrations (116).

Recently, the use of SpA-LacI and SpA-streptavidin fusion proteins in various immunodetection techniques was reviewed (3). Using sandwich techniques, DNA fragments also can be involved in various purification and detection strategies (Fig. 5). Taking advantage of the lac repressor (LacI) from E. coli, binding to the lac operator sequence, a LacI-SpA fusion protein was produced (117) for simplified analysis of polymerase chain reaction (PCR) amplified DNA fragments generated for diagnosis of pathogens (118) (Fig. 5a). In addition, this LacI-SpA fusion protein has been used for mild reversible recovery of protein antigens or whole cells using paramagnetic beads with a coupled DNA fragment containing the lacO recognition sequence (119) (Fig. 5b).

An antigen detection system, termed immuno-PCR, has been described (120) in which the principle of immunode-tection was combined with the sensitivity of PCR. A streptavidin-SpA chimera was used to attach a biotiny-lated DNA fragment to antigen-IgG complexes immobilized on microtiter plate wells (Fig. 5c). Although the method gives a sensitivity greater than any existing antigen-antibody detection system, the sensitivity of the PCR assay could have the drawback of causing false positives resulting from nonspecific binding of IgG, fusion protein, or DNA. Nevertheless, SpA fusion proteins have through the IgG Fc binding activity found extensive use in a vast number of immunodetection techniques.

Protein G Fusion Used to Accomplish "Hot-Start PCR"

Immobilization of enzymes has proven to be a useful procedure for many applications because of the altered characteristics of the immobilized enzyme as compared with the enzyme's soluble counterpart (121). One practical application utilizing such differential activities was recently described in which a heat-mediated release of an affinity-immobilized recombinant Taq DNA polymerase (TagTaq) was used to create a hot-start PCR procedure. Taq DNA polymerase was produced in E. coli as fused to the multifunctional Bio-His6-ABP tag (see foregoing text; 122) for affinity purification and immobilization. HSA-affinity immobilization of the fusion employing the ABP moiety resulted in an apparent deactivation of the Taq DNA poly-merase. However, the ABP-HSA interaction was shown to

IgG-Sepharose

SpA-LacI

IgG-Sepharose

SpA-LacI

Biotinylated DNA fragment

Streptavidin-AlkPho lacO (a)

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