The availability of soluble recombinant proteins has become a necessity of the modern research laboratory. Soluble Fc fusion proteins have a number of uses within the laboratory. As shown in Table 1, these Fc fusion proteins can be useful for in vitro as well as in vivo investigations. To facilitate the manufacture and testing of soluble Fc fusion proteins, this chapter will describe in detail the experimental methods and materials required for generating Fc fusion proteins containing a recombinant protein of interest.
A major advantage of generating Fc fusion proteins is the ability of the investigator to produce specific moieties of any protein. In order to generate
From: Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ
Summary of Fc Fusion Protein Applications
Field of interest
Biochemical Flow cytometry and applications immunohistochemistry
Phage or yeast display
Domain specificity studies
In vitro assays
Soluble P/Fc can be used 10
in vivo to determine therapeutic outcome Animals can be immunized with P/Fc to create polyclonal or monoclonal antibodies Various clearance and half-life studies may be performed with soluble P/Fc fusion proteins Biotinylated P/Fc can be used to detect other proteins of interest P/Fc may be used as a source of antigen (Ag) in order to generate fully human antibodies Mutant proteins can be 17
produced in order to study significant moieties within the protein P/Fc can be used to 18,19
antagonize or agonize cell surface receptor-ligand interactions
full-length Fc fusion proteins, the N-terminus of the protein of interest containing the biologically active site is linked to murine Fc (hinge-CH2-CH3). To describe the orientation of the protein linked to the N-terminus of murine Fc, it is designated as P/Fc. The purpose for fusing murine Fc to the protein of interest is that it allows for easy purification and also increases the half-life of the Fc fusion protein in vivo, which may be useful in immunotherapeutic applications.
Another major advantage of generating Fc fusion proteins using the method described here is that it allows the investigator to produce the Fc fusion protein in large scale (milligram-to-gram quantities). This provides the investigator with large amounts of stable protein sufficient for an extended period of time, which may aid the investigator in reproducing various datasets.
As shown in Table 1, a significant application of Fc fusion proteins is that they may be used as a source of antigen for the generation of either murine or fully human antibodies that may be too expensive to buy commercially, especially if animal studies are planned. Moreover, soluble Fc fusion proteins may be used for in vivo immunizations of mice in order to produce monoclonal antibodies using standard hybridoma techniques (1-3). In addition, advancements in antibody (Ab) technology have led to the creation of synthetic libraries, which enable the investigator to generate fully human antibodies against any protein moiety of interest. For example, a recent advancement led to the construction of a yeast library comprised of yeast that express a wide repertoire of human Ab fragments on the cell surface (4,5). The generation of soluble Fc fusion proteins allows the investigator to utilize such technology in order to quickly analyze and obtain human Ab fragments, which are specific for the protein moiety.
In addition, the investigator may use the soluble Fc fusion protein to detect other target proteins of interest. The Fc fusion proteins can easily be biotinylated for detection by streptavidin conjugates for either flow cytometry or immuno-histochemistry. Also, obtaining a variety of biotinylated Fc fusion proteins would allow the investigator to detect many target proteins at the same time. Therefore, biotinylated Fc fusion proteins may substitute for an Ab that may not be commercially available to detect the target protein of interest.
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