Composite

Part:BBa_K5271011

Designed by: Yin Yan Chan   Group: iGEM24_HKPOLYU   (2024-09-30)
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HER2nb-Fc-EGFRnb

A nanobody composed of two binding regions of EGFR and HER2 linked by a fragment of Fc region of the heavy chain of human IgG.


Profile

  • Name: HER2nb-Fc-EGFRnb
  • Base Pairs: 1515 bp
  • Amino acid: 504 a.a
  • Origin: Synthetic
  • Property: A dual targeting nanobody with a molecular weight of 55 kDa consisting of a Fc fragment and two HER2 and EGFR nanobody binding domains that specifically bind to EGFR and HER2.



Usage and Biology

We used AlphaFold to create a three-dimensional model of HER2nb-Fc-EGFRnb. (Figure 1.) [Jumper et al., 2021] We could clearly identify the two distinct HER2 and EGFR binding domains (Green & Orange), which are spatially separated and connected by by a Fc fragment (Blue) in the predicted 3D structure of our part.


her2-fc-egfr-structure1.png
Figure 1. The AlphaFold predicted structure of HER2-Fc-EGFR. Red: His-tag; Green: HER2 nanobody; Grey: linker; Blue: Fc protein; Orange: EGFR nanobody.




Expression

The recombinant HER2nb-Fc-EGFRnb was sub-cloned into pET-24d(+) expression vector. The pET-24d(+)-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 ~55 kDa.



sds-gel-her2-fc-egfr-1.png
Figure 2. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total 30℃ HER2-FcC-EGFR in 6hr uninduced fraction; lane 3, Total 30℃ HER2-FcC-EGFR in 6hr 0.5mM IPTG induced fraction; lane 4, Total 30℃ HER2-FcC-EGFR overnight uninduced fraction; lane 5, Total 30℃ HER2-FcC-EGFR overnight 0.5mM IPTG induced fraction; lane 6, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 7, Total 16℃ HER2-FcC-EGFR overnight uninduced fraction; lane 8, Total 16℃ HER2-FcC-EGFR overnight 0.5mM IPTG induced fraction. ON = overnight.​



sds-gel-her2-fc-egfr-2.png
Figure 3. Protein expression of HER2-FC-EGFRnb. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total 25℃ HER2-FC-EGFR-1 in 6hr uninduced fraction; lane 3, Total 25℃ HER2-FC-EGFR-1 in 6hr 0.5mM IPTG induced fraction; lane 4, Total 25℃ HER2-FC-EGFR-1 overnight uninduced fraction; lane 5, Total 25℃ HER2-FC-EGFR-1 overnight 0.5mM IPTG induced fraction; lane 6, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 7, Total 25℃ HER2-FcC-EGFR-2 in 6hr uninduced fraction; lane 8, Total 25℃ HER2-FcC-EGFR-2 in 6hr 0.5mM IPTG induced fraction; lane 9, Total 25℃ HER2-FcC-EGFR-2 overnight uninduced fraction; lane 10, Total 25℃ HER2-FcC-EGFR-2 overnight 0.5mM IPTG induced fraction. ON = overnight, hr= Hours



sds-gel-her2-fc-egfr-3.png
Figure 4. Protein expression of HER2-FcC-EGFRnb. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Soluble 25℃ HER2-FcC-EGFR-1 in 6hr 0.5mM IPTG induced soluble fraction; lane 3, Insoluble 25℃ HER2-FcC-EGFR-1 in 6hr IPTG induced insoluble fraction; lane 4, Soluble 25℃ HER2-FcC-EGFR-1 overnight IPTG induced soluble fraction; lane 5, Insoluble 25℃ HER2-FcC-EGFR-1 overnight IPTG induced insoluble fraction; lane 6, Soluble 25℃ HER2-FcC-EGFR-2 in 6hr IPTG induced soluble fraction; lane 7, Insoluble 25℃ HER2-FcC-EGFR-2 in 6hr IPTG induced insoluble fraction; lane 8, Soluble 25℃ HER2-FcC-EGFR-2 overnight IPTG induced soluble fraction; lane 9, Insoluble 25℃ HER2-FcC-EGFR-2 overnight IPTG induced insoluble fraction; lane 10, Soluble 25℃ HER2-FcC-EGFR-2 in 6hr uninduced soluble fraction. ON = overnight; Sol = soluble; Insol = insoluble​



sds-gel-her2-fc-egfr-4.png
Figure 5. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 3, Total HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 4, Total HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 5, Total HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 6, Total HER2-FcC-EGFR in 25℃ with 0.2mM IPTG induced fraction; lane 7, Total HER2-FcC-EGFR in 25℃ uninduced fraction; lane 8, Insoluble HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 9, Insoluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 10, Insoluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction. Insol = insoluble​




sds-gel-her2-fc-egfr-5.png
Figure 6. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Soluble HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 3, Soluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 4, Soluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 5, Soluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 6, Soluble HER2-FcC-EGFR in 25℃ with 0.2mM IPTG induced fraction; lane 7, Soluble HER2-FcC-EGFR in 25℃ uninduced fraction; lane 8, Insoluble HER2-FcC-EGFR in 16℃ with 0.2mM uninduced fraction; lane 9, Insoluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 10, Insoluble HER2-FcC-EGFR e in 25℃ with 0.2mM uninduced fraction. Sol=soluble; Insol = insoluble


As seen from the above results, we could not observe any soluble form of HER2nb-Fc-EGFRnb but insoluble form from two different colonies (HER2-Fc-EGFR-1 & HER2-Fc-EGFR-2). This was probably due to the formation of inclusion bodies inside the cytoplasm and the poor secretion of the protein caused by the hydrophobic Fc fragment. As a result, we could not successfully express the HER2nb-Fc-EGFRnb in our 1st phase of engineering cycle. We further changed the part design by incorporating a secretory signaling peptide unique for E. Coli in front of the HER2nb-Fc-EGFRnb coding sequence, aiming to enhance proper folding of the nanobody and increase the periplasm secretion in our 2nd engineering cycle.




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.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 154
    Illegal NheI site found at 1615
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 643
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 820
    Illegal AgeI site found at 1275
    Illegal AgeI site found at 1407
  • 1000
    COMPATIBLE WITH RFC[1000]


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