Part:BBa_K5271005

Fc fragment

A fragment of Fc region in the heavy chain of human IgG connecting EGFR and HER2 nanobody.

Profile

• Name: Fc fragment for Panobody
• Base Pairs: 702 bp
• Amino acid: 234 a.a
• Origin: Homo sapiens
• Property: A fragment of Fc region in the heavy chain of human IgG connecting EGFR and HER2 nanobody.

Usage and Biology

This part encodes a partial fragment of the Fragment of crystallizable (Fc) region in the heave chain of human Immunoglobin gamma (IgG) [Kulemzin et al., 2017]. It is a 234 amino acid long Fc fragment with a molecular weight of 26.3 kDa. The primary function of this Fc fragment is to link and separate the two binding domains of EGFR and HER2 of our team’s nanobody. Owing to the intrinsic limitations, such as the lack of Fc region to initiation effector functions through antibody dependent cytotoxicity (ADCC) as well as the low serum stability and persistence [Jin et al., 2023], in the first phase of our engineering cycle, we fused a partial Fc fragment to our nanobody design aiming to enhance the potency of the nanobody and trigger Fc-mediated toxicities through ADCC. [Bao et al., 2021] Moreover, the fusion of Fc region to nanobody can further enhance the nanobody stability and prolong the half life of nanobody in vivo. However, in line with Klint et al., our wet lab expression data suggested that the Fc fragment is unfavorable for secretion of Fc protein to periplasm space using prokaryotic expression system such as E. coli.

Source

The DNA sequence of the Fc fragment was obtained from part of the Fc region of the heavy chain of immunoglobin gamma (IgG) of human.

Design Note

Avoid fusing Fc fragment to nanobody when using a prokaryotic system. Insufficient secretion and formation Inclusion bodies were found when expressing the Fc-fused nanobody in E. Coli.

Reference

• Kulemzin, S. V., Chikaev, N. A., Volkova, O. Y., Kuznetsova, V. V., Taranin, A. V., & Gorchakov, A. A. (2017). Modular lentiviral vectory system for optimization of chimeric antigen receptor design. Russ J Bioorganic Chem, 43, 1-9.
• Jin, B. K., Odongo, S., Radwanska, M., & Magez, S. (2023). NANOBODIES®: A Review of Generation, Diagnostics and Therapeutics. International journal of molecular sciences, 24(6), 5994.
• Bao, G., Tang, M., Zhao, J., & Zhu, X. (2021). Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI research, 11, 1-13.
• Klint, J. K., Senff, S., Saez, N. J., Seshadri, R., Lau, H. Y., Bende, N. S., ... & King, G. F. (2013). Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PloS one, 8(5), e63865.
• De Marco, A. (2020). Recombinant expression of nanobodies and nanobody-derived immunoreagents. Protein expression and purification, 172, 105645.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 165
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 342
  
• 1000
  COMPATIBLE WITH RFC[1000]