Latest revision as of 08:18, 2 October 2024

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).

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.

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.​

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

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Sequence and Features