Characterization of the Fc–III–4C-based recombinant protein expression system by using carbonic anhydrase as the model protein

• Published in: Protein expression and purification Vol. 177; p. 105761
• Main Authors: Gong, Yiyi, Yi, Meiqi, Zhang, Lin, Feng, Shan, Deng, Haiteng
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021
• Subjects: Cyclic peptide development and application; Promotion of enzyme activity by fusion tag; Protein expression and purification; Cyclic peptide development and application; Promotion of enzyme activity by fusion tag; Protein expression and purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2020.105761

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. A Scheme illustrates that Fc–III–4C tagged CA can be purified by IgG-immobilized agarose beads. [Display omitted] •The recombinant expressed Fc-III-4C tag strongly binds to the Fc region of IgG, even stronger than protein A/G.•Targeted proteins, fused with Fc-III-4C tag, can be easily purified with IgG-immobilized beads.•Fc-III-4C can boost enzyme activities by trapping transition metal ions using carbonic anhydrase (CA) as the model.