Composite

Part:BBa_K5271012

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


sec-HER2nb-Fc-EGFRnb

A nanobody of HERnb-Fc-EGFRnb carrying a secretory peptide for improved secretion



Profile

  • Name:sec-HER2nb-Fc-EGFRnb
  • Base Pairs: 1581 bp
  • Amino acid: 527 a.a
  • Origin: Synthetic
  • Property: A dual targeting nanobody with a molecular weight of 57.5 kDa consisting of a secretory peptide, Fc fragment and two HER2 and EGFR nanobody binding domains that specifically bind to EGFR and HER2.



Usage and Biology

Based on our primary design in 1st engineering cycle, our team added a secretory peptide in front of the coding sequence of nanobody containing the Fc fragment in attempt to solve the problem of insolubility and the formation of inclusion body. We also created the 3D structure of this part by AlphaFold. Similarly to the HER2nb-Fc-EGFRnb, there are two distinct HER2 and EGFR binding domains (Green & Orange) connected by Fc fragment (Blue) and the secretory signal peptide (Cyan).



sec-her2-fc-egfr-structure1.png
Figure 1. The AlphaFold predicted structure of sec-HER2-Fc-EGFR. Cyan: secretory signal peptide ssSTII1 S-13L; Red: His-tag; Green: HER2 nanobody; Grey: linker; Blue: Fc protein; Orange: EGFR nanobody.




Expression

The recombinant sec-HER2nb-Fc-EGFRnb was sub-cloned into pET-24d(+) expression vector. The pET-24d(+)-sec-HER2nb-Fc-EGFRnb was transformed into Bl21 (DE3) competent cells. Following induction with IPTG, bacterial cells were cultured at 16°C, 25°C and 30°C, respectively, overnight (Figure 2 - 4). Harvested cells were lysed, and the total cell lysate (referred to as the "Total" fraction) was collected. Post-centrifugation, the supernatant (referred to as the "Soluble" fraction) was isolated. Both total and soluble samples were denatured by heating at 100°C for 5 minutes in the presence of 5X SDS loading buffer. These samples were subjected to SDS-PAGE electrophoresis. The target protein, HER2nb-Fc-EGFRnb, was expected to have a molecular weight (MW) of ~57.5 kDa.



sds-gel-sec-her2-fc-egfr-1.png
Figure 2. Protein expression of sec-HER2-Fc-EGFR. Lane 1, Total sec-HER2-Fc-EGFR in 16℃ with 0.5mM IPTG induced fraction for 24 hours; lane 2, Total sec-HER2-Fc-EGFR in 25℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Total sec-HER2-Fc-EGFR in 30℃ with 0.5mM IPTG induced fraction for 24 hours; lane 4, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 5, Total sec-HER2-Fc-EGFR in 16℃ with uninduced fraction for 24 hours; lane 6, Total sec-HER2-Fc-EGFR in 25℃ with uninduced fraction for 24 hours; lane 7, Total sec-HER2-Fc-EGFR in 30℃ with uninduced fraction for 24 hours.​



sds-gel-sec-her2-fc-egfr-2.png
Figure 3. Protein expression of sec-HER2-Fc-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Soluble sec-HER2-Fc-EGFR in 16℃ with 0.5mM IPTG induced fraction for 24 hours; lane 3, Soluble sec-HER2-Fc-EGFR in 25℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Soluble sec-HER2-Fc-EGFR 5 in 30℃ with 0.5mM IPTG induced fraction for 24 hours; lane 5, Soluble sec-HER2-Fc-EGFR 5 in 16℃ with uninduced fraction for 24 hours; lane 6, Soluble sec-HER2-Fc-EGFR in 25℃ with uninduced fraction for 24 hours; lane 7, Soluble sec-HER2-Fc-EGFR in 30℃ with uninduced fraction for 24 hours.​



sds-gel-sec-her2-fc-egfr-3.png
Figure 4. Protein expression of sec-HER2-Fc-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Insoluble sec-HER2-Fc-EGFR in 16℃ with 0.5mM IPTG induced fraction for 24 hours; lane 3, Insoluble sec-HER2-Fc-EGFR in 25℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Insoluble sec-HER2-Fc-EGFR 5 in 30℃ with 0.5mM IPTG induced fraction for 24 hours; lane 5, Insoluble sec-HER2-Fc-EGFR 5 in 16℃ with uninduced fraction for 24 hours; lane 6, Insoluble sec-HER2-Fc-EGFR in 25℃ with uninduced fraction for 24 hours; lane 7, Insoluble sec-HER2-Fc-EGFR in 30℃ with uninduced fraction for 24 hours.​


Unexpectedly, the addition of the secretory peptide did not solved the insolubility problem. There was no soluble form of the nanobody was observed, and almost all protein was expressed as insoluble form. Even the addition of the secretory signal peptide, it could not improve the secretion and proper folding of the nanobody. Owing to this observation, we speculated that the Fc fragment was probably too hydrophobic for proper folding and secretion. As a result, we simplified the structure by removing the Fc fragment, leaving a GSSG linker connecting the two binding domain in our 3rd engineering cycle. This attempted to reduce the complexity of protein for better folding and solubility.




Reference

  • 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.


Sequence and Features


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]


[edit]
Categories
//awards/composite_part
//proteindomain/binding
Parameters
None