Composite

Part:BBa_K5271012:Design

Designed by: Yin Yan Chan   Group: iGEM24_HKPOLYU   (2024-09-30)


sec-HER2nb-Fc-EGFRnb


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 228
    Illegal NheI site found at 1689
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 717
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 894
    Illegal AgeI site found at 1349
    Illegal AgeI site found at 1481
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The secretory sequence has mild to insignificant effect in facilitating nanobody with humanized Fc fragment. The nanobody containing this secretory peptide failed to folded into soluble form and secrete to the periplasm space.

The VHH domain of the nanobody was isolated from a phage library generated from Llama glama lymphocytes that had been immunized with A431 epidermoid carcinoma cells [Roovers et al., 2007]

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.

The nanobody is an antigen-binding fragments that are derived from Camelus dromedarius heavy-chain antibodies and have advantageous characteristics compared with mAbs and their derived fragments for in vivo targeting [Hamers-Casterman et al., 1993]


Source

The DNA sequence of secretory peptide is obtained partially from the Heat-Stable Enterotoxin II (ssSTII) of E.Coli.

References

  • Roovers, R. C., Laeremans, T., Huang, L., De Taeye, S., Verkleij, A. J., Revets, H., ... & van Bergen en Henegouwen, P. M. P. (2007). Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR Nanobodies. Cancer immunology, immunotherapy, 56, 303-317.
  • D'Huyvetter, M., De Vos, J., Xavier, C., Pruszynski, M., Sterckx, Y. G., Massa, S., ... & Devoogdt, N. (2017). 131I-labeled anti-HER2 camelid sdAb as a theranostic tool in cancer treatment. Clinical cancer research, 23(21), 6616-6628.
  • Hamers-Casterman C, Atarhouch T, Muyldermans S. Naturally occurring antibodies devoid of light chains. Nature 1993;363:446–48.
  • Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, et al Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 2011;25:2433–2446.
  • 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.
  • McCarthy, J. E., & Gualerzi, C. (1990). Translational control of prokaryotic gene expression. Trends in Genetics, 6, 78-85.
  • Simmons, L. C., & Yansura, D. G. (1996). Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nature biotechnology, 14(5), 629-634.
  • Zhou, Y., Liu, P., Gan, Y., Sandoval, W., Katakam, A. K., Reichelt, M., ... & Reilly, D. (2016). Enhancing full-length antibody production by signal peptide engineering. Microbial Cell Factories, 15, 1-11.