Part:BBa_K5271013

Panobody -scrambled

A nanobody composed of two binding regions with a scrambled amino acid sequence that acts as a control for the dual targeting nanobody -Panobody.

Profile

• Name: Panobody -scrambled
• Base Pairs: 777 bp
• Amino acid: 258 a.a
• Origin: Synthetic
• Properties: A scrambled control for the dual targeting nanobody -Panobody

Usage and Biology

To verify our design, we used Alphafold to create a three-dimensional model of Panobody -scrambled (Figure 1). [Jumper et al., 2021] Subsequently, we perform a molecular docking analysis to examine the binding affinity of the scrambled control.

Figure 1. The AlphaFold predicted structure of Nanobody scramble. Red: His-tag; Pink: Nanobody scramble; Grey: linker.

The molecular docking analysis of Panobody- scrambled revealed a less extensive and intricate binding network. The scrambled HER2 binding region of Panobody- scrambled interacts with significant residues of the HER2 receptor, such as Lys765, Thr759, and Ash838, forming key hydrogen bonds and salt bridges and π-π stacking interactions, with Tyr835 playing a stabilizing role (Fig. 2a). However, the overall interaction network was less comprehensive than that observed for the Panobody. Similarly, when the Panobody -scrambled bind to EGFR receptor, it showed that it forms crucial hydrogen bonds and salt-bridge interactions with key residues (Lys739, Val769, Asp770, Hie773, Lys823, and Thr847) of the EGFR receptor (Fig. 2b); however, the binding appears less reinforced, as hydrogen bonds are not as widespread or integrative as in the Panobody-EGFR complex.

Figure 2. (a) 3D and 2D visualizations of molecular docking between the HER2 binding region of Panobody -scrambled the HER2 receptor. (b) 3D and 2D visualizations of molecular docking between the EGFR binding region of Panobody -scrambled and the EGFR receptor.

Expression

The recombinant Panobody -scrambled is sub-cloned into pET-24d(+) expression vector. The pET-24d(+)-Panobody -scrambled was transformed into Bl21 (DE3) competent cells. The positive colony was picked and grow. Following induction with 0.5 mM IPTG, bacterial cells were cultured at 16°C, 25°C and 30°C, respectively, overnight (Figure 3 - 5). 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, Panobody, was expected to have a molecular weight (MW) of ~27.9 kDa.

Figure 3. Protein expression of Panobody -scrambled. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total scrambled Panobody in 16℃ with 0.5mM IPTG induced fraction for 24 hours; lane 3, Soluble scrambled Panobody in 16℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Insoluble scrambled Panobody in 16℃ with 0.5mM IPTG induced fraction for 24 hours; lane 5, Total scrambled Panobody in 16℃ with uninduced fraction for 24 hours; lane 6, Soluble scrambled Panobody in 16℃ with uninduced fraction for 24 hours; lane 7, Insoluble scrambled Panobody in 16℃ with uninduced fraction for 24 hours.

Figure 4. Protein expression of scrambled Panobody. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total scrambled Panobody in 25℃ with 0.5mM IPTG induced fraction for 24 hours; lane 3, Soluble scrambled Panobody in 25℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Insoluble scrambled Panobody in 25℃ with 0.5mM IPTG induced fraction for 24 hours; lane 5, Total scrambled Panobody in 25℃ with uninduced fraction for 24 hours; lane 6, Soluble scrambled Panobody in 25℃ with uninduced fraction for 24 hours; lane 7, Insoluble scrambled Panobody in 25℃ with uninduced fraction for 24 hours.​

Figure 5. Protein expression of scrambled Panobody. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total scrambled Panobody in 30℃ with 0.5mM IPTG induced fraction for 24 hours; lane 3, Soluble scrambled Panobody in 30℃ with 0.5m IPTG induced fraction for 24 hours; lane 3, Insoluble scrambled Panobody in 30℃ with 0.5mM IPTG induced fraction for 24 hours; lane 5, Total scrambled Panobody in 30℃ with uninduced fraction for 24 hours; lane 6, Soluble scrambled Panobody in 30℃ with uninduced fraction for 24 hours; lane 7, Insoluble scrambled Panobody in 30℃ with uninduced fraction for 24 hours.​

However, there was no soluble Panobody -scrambled found from the above expression conditions. Panobody -scrambled was unable to proceed to purification.

Source

The sequence of the Panobody -scrambled was randomly generated and had a same length with the Panobody.

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.
• Schmitz, K. R., Bagchi, A., Roovers, R. C., en Henegouwen, P. M. V. B., & Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7), 1214-1224.
• 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.
• Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., ... & Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. nature, 596(7873), 583-589.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 834
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]