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Updated: Dec 8, 2025
Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Characterization of the Fc-III-4C-based recombinant protein expression system by using carbonic anhydrase as the
Yiyi Gong1, Meiqi Yi2, Lin Zhang3
1Medical Research Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China; MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systematic Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Abstract:
Development of new affinity tags is important for recombinant protein expression and purification. Based on our earlier work, we devised an affinity tag by addition of two cysteine residues onto the N- and C-termini of the Fc-III peptide and designated as the Fc-III-4C tag, in which four cysteine residues form two disulfide linkages. The binding affinity of Fc-III-4C tag to human IgG is measured as 2.28 nM (Kd) and is 100 times higher than that of the Fc-III tag to IgG. Fc-III-4C tagged carbonic anhydrase (CA) can be effectively purified with IgG-immobilized beads, and Fc-III-4C tag does not possess adverse effects on the structure and stability of CA. Furthermore, the Fc-III-4C tagged protein binds to multiple transition metal ions, which enhances activities of enzymes that use metal ions as co-factors. These results suggest that Fc-III-4C tag is a useful tool for expression and purification of recombinant proteins and enhances the activities of some fusion proteins that use Zn2+ or Cu2+ as cofactors.
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