Biotinylated DNA fragment for PCR amplification

Streptavidin-SpA

Antigen

Streptavidin-SpA

Antigen

Figure 5. Schematic presentation of some sandwich concepts utilizing SpA fusion proteins and interacting DNA fragments for detection and purification purposes. (a) Colorimetric detection of DNA fragments containing the lac operator (lacO) sequence, using a lac repressor (LacI)-SpA fusion protein immobilized to IgG-Sephar-ose. The colorimetric signal was obtained using a strep-tavidin-alkaline phosphatate conjugate. The DNA fragments, generated by the PCR technique, introduce biotin and the lac operator by the PCR primers. This assay has been used to detect Plasmodium falciparum DNA in clinical samples (118). (b) This system takes advantage of the same interactions as described in a, with the only difference that paramagnetic beads with covalently coupled streptavidin are used as solid support. The technique has been used to purify different proteins and also whole cells using specific antibodies reversibly immobilized to the paramagnetic beads via the LacI-SpA fusion and a biotinylated DNA fragment. Mild recovery of the antigen-antibody complexes can be achieved using deoxyribonuclease (DNase) or restriction endonucleases (119). (c) In this system, the antigen, immobilized on a microtiter plate, is allowed to react with a monoclonal antibody, linked to a biotinylated DNA fragment via an SpA-streptavidin chimera. The detection is accomplished by PCR amplification using primers specific for the bound DNA. The generated fragment is analyzed by agarose gel electrophoresis (120).

be disrupted at elevated temperatures (>70 °C), resulting in a restored DNA polymerase activity of released Taq DNA polymerase fusion protein. This concept for controlling the activity of the Taq DNA polymerase was demonstrated to be of practical use to achieve a so-called hot-start PCR procedure, where it is desired to suppress the DNA polymerase activity until temperatures are reached at which nonspecific annealing of PCR primers is eliminated. The affinity immobilization concept for hot-start PCR was successfully used in full multiple-cycle PCR amplification and was shown to eliminate artefactual primer-dimer products in the amplification of the human oncogene K-ras gene in contrast to standard amplification protocols. Noteworthy, the ABP affinity fusion partner was still functional after 30 PCR cycles, allowing post-PCR reimmobilization of the Taq DNA polymerase fusion protein onto fresh HSA-Sepharose, facilitating the removal of the DNA polymerase from reaction mixtures after PCR (122). Further studies are needed to investigate if this concept for modulating biological activity can also be applied to other widely used enzymes, for example, reverse transcriptase.

Immunogen-encoding genes

Clone into appropriate ZZ-fusion vector

E. coli expression x

IgG-purification

Clone into appropriate BB/ABP-fusion vector

E. coli expression x

HSA-purification

Immunization Analysis of immune responses

Efficacy studies with affinity-purified antigen-specific antibodies

Protein A and Protein G Fusion Proteins in Subunit Vaccine Development

Immunogenic proteins and peptides that are produced as fusion proteins have been frequently used for immunization purposes and represent a strategy to construct subunit vaccines against infectious diseases. An important aspect regarding production of antigens by recombinant DNA technology is the purification of the gene product from contaminating host components. The approach to express peptides or proteins as one part of a fusion protein, where the other part constitutes an affinity handle, allows rapid and efficient affinity purification of the gene product.

As a powerful strategy in subunit vaccine development, a dual expression system was devised, combining the two similar affinity-fusion expression systems (ZZ and BB) for the parallel expression of immunogenic peptides and proteins (Fig. 8) (50). After affinity purification, one fusion protein is used for immunization while the corresponding second fusion protein is used to analyze the induced antibody response to the fused peptide. This strategy eliminates any background originating from antibodies reactive with the affinity tag of the fusion used for immunization. In addition, the second fusion protein can, after immobilization, be used as a ligand for affinity purification of peptide-specific antibodies (Fig. 8). The dual expression system has been used to produce immunogenic structures derived from different Plasmodium falciparum malaria blood-stage antigens, and the expressed fusion proteins have been shown to be immunogenic in mice, rabbits, and monkeys and to induce antigen-specific antibody and T-cell responses (for review, see Ref. 27). The dual expression strategy has thus shown to be of significant value in subunit vaccine research.

Besides the use of BB and ABP fusion proteins in the dual expression concept for analysis of elicited immune responses, the serum albumin-binding region of SpG has been shown to have inherent immunopotentiating properties when used as a carrier protein genetically fused to

Figure 6. A flow chart representation of how the dual expression concept has been used in subunit vaccine research. Note that the BB-P fusion protein can be used both as coating antigen in various assays to analyze elicited immune responses and also in an immobilized form for enrichment of antigen-specific antibodies from the immune sera. The use of this concept has been thoroughly reviewed (27).

the immunogen used for immunization (27,49,123,124). Recently, it was demonstrated that a BB-fusion protein (BB-M3), containing a malaria peptide, induced significant antibody responses in mice strains that were nonrespon-ders to the malaria peptide (M3) alone, suggesting that BB has the ability to provide T-cell help for antibody production (49). Furthermore, a fusion protein BB-G2N, comprising a 101-amino acid sequence from human RSV, was shown to induce protective immunity in mice to RSV challenge (123,124). It was shown that, by inclusion of the BB part, a more potent G2N-specific B-cell memory response was evoked (124). This indicates that the SpG-derived BB can function both as an affinity tag to facilitate purification and as a carrier protein with immunopotentiating properties. To date, it is not fully elucidated whether this capacity results from strong T-cell epitopes (49) or is related to the serum albumin-binding activity resulting in a prolonged exposure of the immunogen to the immune system, or is due to a combination of both.

Protein A and Protein G Fusion Proteins in Functional cDNA Analysis

The principle of dual expression systems was recently also implemented for functional analysis of cDNA-encoded proteins (Fig. 7) (13). Selected cDNAs were then expressed as ZZ fusion proteins and used for immunization to elicit antibodies, whereas the corresponding ABP fusion protein, having the cDNA-encoded portion in common, was used for purification of target protein-specific antibodies. Such

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