Development of the Fc-III Tagged Protein Expression System for Protein Purification and Detection

Abstract

In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from E. coli. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG-Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox.

Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

“…The Fc-III tag is the newest fusion tag that can be used for protein purification and detection. 10 The Fc-III peptide in expressed recombinant proteins is cyclized through a disulfide bond that tightly binds to recombinant IgG-Fc expressed in E. coli. Immobilized IgG or IgGFc can be used to purify recombinant Fc-III-tagged proteins from cell lysates.…”

“…The gene of human MM-CK and the pET-21a expression vector were used for His-tagged and the Fc-III-tagged CK construction as described in the early report. 10 Protein expression and purification GFP and the Fc-III-tagged GFP were expressed in 293T cell line that was purchased from the American Type Culture Collection. Using Lipofect 2000 transfection reagent from Invitrogen (Carlsbad, CA), all plasmids were transfected into 293T cell line, which was then cultured over 24 hours.…”

“…10 Using the Fc-III tag, we purified the Fc-III-tagged carbonic anhydrase and detected the Fc-III-tagged CD38 on cell surface. The Fc-III tag has the potential to become a useful tag for protein purification and detection.…”

“…The Fc-III tag, which contains 13 amino acid residues (DCAWHLGELVWCT), is a newly developed affinity tag. 10 It mimics the binding of Protein A or Protein G to the hinge region of Fc fragment of IgG molecules. 11,12 We showed that the peptide was cyclized by the disulfide linkage between Cys2 and Cys12 when the Fc-III-tagged protein was expressed, and the Fc-III-tagged proteins tightly bind to recombinant IgG-Fc expressed in Escherichia coli…”

“…11,12 We showed that the peptide was cyclized by the disulfide linkage between Cys2 and Cys12 when the Fc-III-tagged protein was expressed, and the Fc-III-tagged proteins tightly bind to recombinant IgG-Fc expressed in Escherichia coli that does not have glycosylation. 10 Using the Fc-III tag, we purified the Fc-III-tagged carbonic anhydrase and detected the Fc-III-tagged CD38 on cell surface. The Fc-III tag has the potential to become a useful tag for protein purification and detection.…”