Fc-Fusion Construction

pFUSE-Fc plasmids contain two innovative composite promoters, EF1 prom / HTLV 5’UTR and CMV enh / FerL prom, providing the following features:
- High levels of expression.
- Expression independent of the cell cycle.
- Transfectable in a variety of mammalian cells, including cells commonly used in protein purification systems (CHO, COS, HEK293).

pFUSE-Fc plasmids are selectable with Zeocin™ in E. coli and mammalian cells. They can be used for transient or stable transfection.

Fc-Fusion Proteins are secreted
Two versions of pFUSE-Fc exist to allow secretion of the Fc fusion:
• pFUSE-Fc1 is recommended when the protein of interest is naturally secreted and thus contains a native signal sequence.
• pFUSE-Fc2 contains the IL2 signal sequence (IL2ss) for the generation of Fc-Fusion proteins derived from proteins that are not naturally secreted.

Ease of Detection
Fc-fusion proteins can be easily detected in the supernatant of pFUSE-Fc-transfected cells by SDS-PAGE. Their functional domains can be identified by immunoblotting and ligand blotting.

Ease of Purification
Most Fc-fusion proteins can be easily purified by single-step protein A or protein G affinity chromatography.

pFUSE-Fc plasmids feature a large choice of Fc regions from different species, with or without introns, native or engineered.

Fc regions from various species
• Human Fc: IgG1, IgG2, IgG3, IgG4
• Mouse Fc: IgG1, IgG2a, IgG2b, IgG3
• Rabbit Fc: IgG
• Rat Fc: IgG2b

Fc regions with introns
pINFUSE-Fc is a new family of pFUSE-Fc plasmids featuring genomic Fc regions. The presence of introns is known to enhance the level of gene expression as splicing is known to promote rapid and efficient mRNA export.

Engineered Fc regions with altered effector functions
• Human IgG1e1 & IgG1e2: increased half-life
• Human IgG1e3 and mouse IgG2Ce1: reduced ADCC and CDC
• Human IgG1e4: increased ADCC and CDC
• Human IgG2e1: reduced CDC

All pFUSE-Fc plasmids are provided as 20 µg of lyophilized DNA.
Product is shipped at room temperature and should be stored at -20°C.
Plasmid is stable up to one year when properly stored.
Each pFUSE-Fc plasmid is provided with 4 pouches of E. coli Fast-Media® Zeo (2 TB and 2 Agar).

Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein that has found application in purifying antibodies through its binding to the Fc region [1].
Protein G binds to most mammalian IgGs through the Fc region, but some binding also occurs through the Fab region.
Native protein G also binds albumin, however, because serum albumin is a major contaminant of antibody sources, the albumin binding site has been removed from recombinant forms of Protein G [2].
Protein G / Agarose from InvivoGen uses the recombinant form of protein G lacking the albumin-binding region.
In Protein G / Agarose, the recombinant protein G is coupled to beads using a leak-resistant chemistry that provides a support with minimal non-specific binding.

1. Sjobring U. et al., 1991. Streptococcal Protein G. Gene structure and protein binding properties. J Biol Chem. 140:1194-1197.
2. Goward CR. et al., 1990. Expression and purification of truncated recombinant streptococccal Protein G. Biochem J. 267:171-177.