Part:BBa_K5271012

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:

Usage and Biology

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

Expression

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.​

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.​

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.​

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.

• 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.

• 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]