Fc-tag Protein Expression and Purification Basics

Fc-tag Basics

What is an IgG-Fc-tag or IgG-Fc chimeric?

IgG-Fc tag is the constant region (domain 3 and 4) of immunoglobulin heavy-chain. It is fused to the C-terminus of a protein and hence it recembles a mouse/human chimeric antibody in a way, and sometimes the Fc-fusion protein is also called Fc chimeric protein. The Fc-tag is about 25 KDa.

Why use an IgG-Fc-tag for protein expression and production?

Adding an IgG-Fc tag to a protein not only allows rapid and simple detection of protein expression by commercially available ELISA kit, but also assist simple affinity purification of the Fc-tagged protein by protein A, protein G, and protein L affinity purification resins. Furthermore, adding an IgG-Fc tag to a protein often increases protein expression yield.

Why IgG-Fc-tagged protein shows as a dimer on non-reduced SDS Page?

Because IgG antibody Heavy chain forms a dimer naturally through the hinge regions cysteines residuals, the IgG-Fc chimeric protein always forms a disulfide-bonded dimer. It shows as a monomer on reduced SDS page, but shows as a homo-dimer on a non-reduced SDS page.

What choices of IgG-Fc can you choose for my Fc-chimeric protein?

You can choose the Fc region of any IgG antibodies from multiple species, depending on your preference of application. Human IgG1-Fc is the most widely used Fc-tag due to easy detection and high binding affinity to protein A resin.

An incomplete list of IgG-Fc for Fc tag is listed below for your reference:
Human: IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc
Mouse: IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG3-Fc
Rat: IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG2c-Fc
Rabbit: IgG-Fc
Canine: IgG-Fc

Fc-tagged Protein Purification

How do you purify Fc-tagged proteins?

Fc-tagged recombinant protein can be affinity purified directly from a cell culture lysate or supernatant. Depending on the selection of the IgG species and the subtype, the Fc-tagged protein binds to the protein A, protein G, or protein L affinity purification resins just like an IgG antibody. After washing away residual impurities, bound Fc-tag proteins can be eluted off the affinity column by low pH buffer. For more information on our high quality and low cost antibody affinity purification resins, please refer to: Protein A Affinity Resin, Protein G Affinity Resin, Protein L Affinity Resin .

How do you cleave of the IgG-Fc-tag after purification?

In some applications, it is desirable to remove the Fc-tag, for example, for protein crystallization. To allow cleavage of the Fc-tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the Fc-tag (Fc-EK site-protein structure) can allow complete removal of the Fc-tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the Fc-tag. For more information on the cleavage site and tag removal by EK and HRV-3C protease, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).

Fc-fusion-tag (Fc-chimeric) Protein Production & Purification Service

Full services From gene cloning/synthesis to purified proteins
Host Cells Choices of expression hosts:
Transient transfection in HEK293 cells
Transient transfection in CHO cells
Stable expression in CHO cells
Yeast cell expression system
Insect cell/baculovirus expression system
Fc Tag Design Fusion with protein at N- or C-terminus
Choices of Fc-tags Human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc
Mouse IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG3-Fc
Rat IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG2c-Fc
Rabbit IgG-Fc
Canine IgG-Fc
Cleavage Site Option to design a cleavage site between the tag and protein
Selection of cleavage protease enzyme: Enterokinase (EK), HRV-3C (human rhinovirus protease), TEV
Purification Affinity Purification by: Protein A Affinity Resin, Protein G Affinity Resin, or Protein L Affinity Resin
Deliverable Purified recombinant proteins (quantities to be specified by customer)
Quality Specification typically >90% purity by SDS page
Typical Timeline 8-12 weeks

IgG-Fc-fusion-tag (Fc-chimeric protein) has been widely used in recombinant protein expression and production. The Fc-fusion-tag allows simple and efficient affinity purification of the tagged protein using protein A or protein G affinity chromatography. The Fc-fusion-tag (Fc-chimeric) also can be used for detection in western blot, ELISA assay by using commercially available antibodies and ELISA kits. The antibody Fc-region is typically fused to the C-terminus of protein and it is mostly used in mammalian cell expression system. The addition of the IgG-Fc could also increase protein solubility and expression yield sometimes. Due to the nature of the antibody IgG-Fc, Fc-chimeric proteins are expressed as homodimer.

Sino Biological has successfully produced many proteins with the antibody IgG-Fc-tag. We are highly experienced in designing the Fc-tag according to the protein structure and handle any problems arising from protein purification process. Sino Biological has also developed its own protein A and protein G affinity purification resin. For more information about our Protein A and Protein G affinity resin, please refer to: Antibody Purification Resins.

Our track-records in the past years on the successful production of about 6,000 recombinant protein bulks and over 12,000 antibodies have attracted top-10 multi-national pharmaceutical customers as well as small biotech companies worldwide through word-of-mouth, as we have not done much business development or advertisement in the past. In addition, we are starting to promote our protein production services to all research institutions and companies worldwide, as we believe more customers can benefit greatly from our capacity and experience.

How do you submit a request for protein production service?

If you are interested in using our protein production services to support your antibody discovery and development program, please contact us by leaving an on-line message or send us your detailed request to CRO-service@sinobiological.com. Based on the quantity and quality requirement of your request, a formal quote will be send back to you for your review, comment, and/or acceptance.

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