Results
Phase I: The Bispecific Nanobody Fc Fusion Protein
1. BioBrick Design
In the initial phase, our goal was to design a bispecific fusion protein drug conjugate targeting both EGFR and HER2, which are upregulated in pancreatic cancer, especially in gemcitabine-resistant cases. We designed a construct consisting of two nanobodies—one for EGFR and one for HER2—linked by a Fc fragment from an antibody. The Fc fragment was expected to enhance the serum stability of the molecule. The plasmid was named pET-24d(+)-HER2-Fc-EGFR (Figure 1).
2. Transformation into TOP10 competent cells
Background:
The recombinant HER2-Fc-EGFR plasmid was digested with XhoI and HindIII restriction enzymes and subsequently ligated to create a functional expression vector. The ligation products were transformed into chemically competent E. coli TOP10 cells, followed by plating on LB agar plates containing kanamycin to select for successful transformants.
Observation:
Multiple bacterial colonies were observed on the LB agar plates, indicating the presence of kanamycin-resistant transformants (Figure 2).
Conclusion:
The transformation of TOP10 cells with the HER2-Fc-EGFR plasmid was successful, as confirmed by the growth of colonies on the kanamycin-containing LB agar plates.
3. Sequencing Result
Background:
Several colonies from the LB agar plates were selected and sent for DNA sequencing to verify the integrity of the HER2-Fc-EGFR construct. The sequencing analysis was aimed at confirming the accuracy of the ligation and checking for any mutations in the coding sequence.
Observation:
No mutations were detected in the coding sequence of the HER2-Fc-EGFR plasmid. The sequence matched the expected construct without deviations.
Conclusion:
The DNA sequencing results confirmed that the HER2-Fc-EGFR recombinant plasmid was correctly assembled and free of mutations, making it suitable for protein expression experiments.
4. Transformation into BL21(DE3)
Background:
The HER2-Fc-EGFR plasmid was further purified and transformed into E. coli BL21(DE3) cells, which are optimized for protein expression. Similar to the TOP10 transformation, only bacteria that successfully incorporated the kanamycin resistance gene survived on the LB agar plates containing kanamycin.
Observation:
Multiple colonies were observed, suggesting successful transformation into BL21(DE3) cells (Figure 3).
Conclusion:
Successful transformation of HER2-fc-EGFR plasmids to BL21(DE3) cells is confirmed by the presence of transformed colonies on the LB agar plate.
5. Protein Expression
Background:
To induce the expression of the HER2-Fc-EGFR fusion protein, BL21(DE3) cells were treated with 0.5 mM IPTG and cultured at different temperatures (16°C, 25°C, and 30°C) overnight. After induction, the cells were lysed using B-PER reagent, and the lysates were separated into soluble (supernatant) and insoluble (pellet) fractions by centrifugation. Both fractions were prepared for SDS-PAGE by mixing with 5X SDS loading buffer and denaturing at 90°C for 5 minutes. The samples were loaded onto an SDS-PAGE gel and run at 120 V for 60 minutes.
Observation:
A distinct band was visible in the lanes containing induced samples. However, the majority of the target protein was found in the insoluble fraction, indicating poor solubility at all tested temperatures. Uninduced samples did not show this band, confirming that the observed band represents the IPTG-induced HER2-Fc-EGFR protein.
Conclusion:
Successful in expressing the protein, but not soluble.
The HER2-Fc-EGFR fusion protein was successfully expressed in E. coli, as confirmed by the distinct band on the SDS-PAGE gels. However, the protein remained largely insoluble, likely due to insufficient solubility under the conditions tested. Future experiments will focus on optimizing the solubility of the expressed protein, potentially by adjusting culture conditions, co-expression with chaperones, or testing different linker regions.
Phase II - Addition of Secretion Signal Peptide
1. BioBrick Design
To further improve the chances of obtaining a soluble protein, we incorporated a secretion signal peptide (ssSTII1 S-13L) to guide the protein to the periplasmic space of E. coli, where folding conditions are more favourable. The signal peptide sequence was introduced before the HER2-Fc-EGFR construct to create the sec-HER2-Fc-EGFR recombinant plasmid, as shown in the vector map (Figure 7).
2. Transformation into TOP10 competent cells
Background:
The sec-HER2-Fc-EGFR recombinant plasmid was digested with XhoI and HindIII and ligated to form a functional plasmid. The ligation products were transformed into chemically competent E. coli TOP10 cells, followed by selection on LB agar plates containing kanamycin. Only bacteria harbouring the kanamycin resistance gene in the recombinant vector were able to survive.
Observation:
Multiple colonies were observed on the LB agar plates, indicating successful transformation (Figure 8).
Conclusion:
The transformation of TOP10 cells with the sec-HER2-Fc-EGFR plasmid was successful, as evidenced by the growth of kanamycin-resistant colonies.
3. Sequencing Result
Background:
To verify the integrity of the recombinant plasmid, colonies were picked from the LB agar plates and sent for DNA sequencing. The sequencing aimed to confirm that the coding sequence of sec-HER2-Fc-EGFR was correctly assembled and mutation-free.
Observation:
Sequencing results showed no mutations in the peptide coding region, confirming the accuracy of the construct.
Conclusion:
The DNA sequencing confirmed that the sec-HER2-Fc-EGFR plasmid was correctly assembled without mutations, making it suitable for protein expression experiments.
4. Transformation into BL21(DE3)
Background:
Following the transformation into TOP10 cells, the sec-HER2-Fc-EGFR plasmid was purified and subsequently transformed into E. coli BL21(DE3) cells for protein expression. Similar to previous transformations, the selection was performed on LB agar plates containing kanamycin.
Observation:
Multiple colonies were observed on the selective LB plates, indicating successful transformation (Figure 9).
Conclusion:
The transformation of BL21(DE3) cells with the sec-HER2-Fc-EGFR plasmid was confirmed by the presence of colonies on the selective plates.
5. Protein Expression
Background:
The sec-HER2-Fc-EGFR fusion protein was expressed in BL21(DE3) cells under different induction conditions. Cells were cultured at 16°C, 25°C, and 30°C with 0.5 mM IPTG for 24 hours to induce protein expression. The protein was extracted using B-PER reagent, and the lysates were centrifuged to separate the soluble (supernatant) and insoluble (pellet) fractions. Both fractions were analyzed by SDS-PAGE. Protein samples were mixed with 5X SDS loading buffer, denatured at 90°C for 5 minutes, and loaded onto an SDS-PAGE gel. Electrophoresis was carried out at 120 V for 60 minutes. The expected molecular weight of the sec-HER2-Fc-EGFR protein was approximately 57.5 kDa.
Observation:
A clear band at ~57.5 kDa, corresponding to the sec-HER2-Fc-EGFR protein, was observed in the induced samples. The uninduced samples lacked this band, confirming IPTG-induced expression. However, the majority of the protein was found in the insoluble fraction, indicating poor solubility even with the addition of the signal peptide (Figures 10,11).
Conclusion:
Successful in expressing the protein, but not soluble.
Although the sec-HER2-Fc-EGFR protein was successfully expressed, it remained largely insoluble under the tested conditions. The presence of the 57.5 kDa band confirmed expression, but the protein's limited solubility suggests further optimization is necessary.
Phase III - Removal of Fc Fragment
1. BioBrick Design
In this final phase, we simplified the construct by removing the Fc fragment entirely, leaving only the two nanobodies targeting EGFR and HER2, joined by a flexible linker. The new design, pET-24d(+)-HER2-EGFR, resulted in a smaller and simpler protein. We named this version Panobody, focusing on the bispecific targeting functionality without the added complexity of the Fc fragment.
2. Transformation into TOP10 competent cells
Background:
The recombinant plasmid pET-24d(+)-Panobody was transformed into TOP10 competent cells. Only bacteria received the Kanamycin-resistant gene in the recombinant vector survived in the LB agar plate containing kanamycin.
Observation:
Multiple colonies were observed on the LB agar plate, indicating successful transformation (Figure 13).
Conclusion:
The successful transformation of the pET-24d(+)-Panobody plasmid into TOP10 cells was confirmed by the presence of colonies on the kanamycin-containing agar plate.
3. Sequencing Result
Background:
Several colonies from the LB agar plates were selected and sent for DNA sequencing to verify the integrity of the pET-24d(+)-Panobody. The sequencing analysis was aimed at confirming the accuracy of the ligation and checking for any mutations in the coding sequence.
Observation:
No mutations were detected in the coding sequence of the pET-24d(+)-Panobody plasmid. The sequence matched the expected construct without deviations.
Conclusion:
The DNA sequencing results confirmed that the pET-24d(+)-Panobody plasmid was correctly assembled and free of mutations, making it suitable for protein expression experiments.
4. Transformation into BL21(DE3)
Background:
The recombinant plasmid pET-24d(+)-Panobody was transformed into Bl21 (DE3) competent cells. Only bacteria received the Kanamycin-resistant gene in the recombinant vector survived in the LB agar plate containing kanamycin.
Observation:
Multiple colonies were observed.
Conclusion:
The presence of colonies on the kanamycin plate confirmed the successful transformation of the pET-24d(+)-Panobody plasmid into BL21 (DE3) cells.
5. Protein Expression
Result of induction at 0.5 mM IPTG and at 16°C:
Background:
Following induction with 0.5 mM IPTG, bacterial cells were cultured at 16°C overnight. 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 at 220 V for 40 minutes. The target protein, Panobody, was expected to have a molecular weight (MW) of ~27.9 kDa.
Observation:
The SDS-PAGE results showed a prominent band at ~27 kDa in the induced sample, especially in the soluble fraction. This band was absent in the uninduced samples, indicating that the protein expression was successfully induced by IPTG. The expected size of the protein aligns with the observed band, as indicated by the red bracket.
Conclusion:
In the IPTG-induced sample, a unique band appears at ~27 kDa, which matches the expected molecular weight of the Panobody. This confirms that the target protein Panobody was successfully expressed under these experimental conditions.
Result of induction at 0.5 mM IPTG and at 25°C
Background:
Following induction with 0.5 mM IPTG, bacterial cells were cultured at 25°C overnight. 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 at 220 V for 40 minutes. The target protein, Panobody, was expected to have a molecular weight (MW) of ~27.9 kDa.
Observation:
The SDS-PAGE results showed a prominent band at ~27 kDa in the induced sample, especially in the soluble fraction. This band was absent in the uninduced samples, indicating that the protein expression was successfully induced by IPTG. The expected size of the protein aligns with the observed band, as indicated by the red bracket.
Conclusion:
In the IPTG-induced sample, a unique band appears at ~27 kDa, which matches the expected molecular weight of the Panobody. This confirms that the target protein Panobody was successfully expressed under these experimental conditions.
Result of induction at 0.5 mM IPTG and at 30°C
Background:
Following induction with 0.5 mM IPTG, bacterial cells were cultured at 30°C overnight. 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 at 220 V for 40 minutes. The target protein, Panobody, was expected to have a molecular weight (MW) of ~27.9 kDa.
Observation:
No bands at ~27 kDa were detected, suggesting a lack of Panobody expression at 30°C.
Conclusion:
No detectable protein bands at the expected molecular weight indicate that Panobody expression was unsuccessful at 30°C. This suggests that the higher temperature may have negatively impacted protein expression or solubility.
6. Purification of protein by Ni-NTA Affinity Chromatography
Background:
In this experiment, the Panobody protein, designed to target both EGFR and HER2, was purified using Ni-NTA affinity chromatography. The binding was facilitated by equilibrating the column with the equilibration buffer, while the washing steps removed non-specifically bound proteins. Finally, an imidazole gradient was used to elute the target Panobody protein, as imidazole competes with histidine residues for binding to nickel, thereby releasing the protein from the column.
First, the column was equilibrated using equilibration buffer to prepare the resin for protein binding. Then, unbound proteins were removed using washing buffer, ensuring that only His-tagged Panobody remained bound to the column. Next, gradient of imidazole was applied to elute the Panobody. Imidazole displaced the His-tagged Panobody from the nickel, leading to the collection of elution fractions. Finally, eluted fractions were collected and mixed with 5X SDS loading buffer. Samples were then denatured by heating at 100°C for 5 minutes. Samples were loaded onto an SDS-PAGE gel and run at 220 V for 40 minutes to assess protein purity and confirm the presence of the target Panobody.
Observation:
(a) FPLC Analysis: Fast protein liquid chromatography (FPLC) results showed significant protein efflux at both 30% and 100% elution phases, suggesting a successful purification of Panobody.
(b) SDS-PAGE Analysis: The SDS-PAGE gel analysis confirmed the presence of the target Panobody protein, with distinct bands observed at approximately ~27 kDa, as highlighted by the red bracket.
Conclusion:
The elution fractions contained a high concentration of Panobody, as confirmed by FPLC and SDS-PAGE analysis. This demonstrates the successful purification of Panobody using Ni-NTA affinity chromatography.
7. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS)
Background:
Following purification, the Panobody protein was buffer-exchanged using Amicon Ultra filtration (10 kDa MWCO) into PBS, to remove imidazole and prepare the sample for further characterization. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) was then performed to confirm the molecular weight of the purified Panobody and validate successful expression and purification.
Observation:
LC-ESI-MS revealed a peak at 27,851 Da, consistent with the expected molecular mass of the Panobody, indicating successful purification and preparation of the protein for downstream applications.
Conclusion:
LC-ESI-MS confirmed the presence of the Panobody in the elution fraction, with a molecular weight of 27,851 Da, validating the effectiveness of the Ni-NTA affinity chromatography.
8. Functional Assay on Panobody
To assess the functional activity of the purified Panobody, a pancreatic cancer cell.
8.1 MTT cytotoxicity confirming Gemcitabine resistance of pancreatic cancer cell lines -PANC1
Background:
(a) Two PANC-1 human pancreatic carcinoma cell lines (mock and gemcitabine resistant GemR) were compared for the gemcitabine resistance.
(b) GemR PanC-1 cells are expected to demonstrate a higher IC50 compared to mock PanC-1 cells.
Observation:
(a) The IC50 for PanC-1 mock and GemR towards gemcitabine were 2.97µM and 11.38µM, respectively.
(b) The GemR cells p is 3.8-fold more resistant than mock control group towards gemcitabine.
Conclusion:
GemR PANC-1 cells demonstrate acquired gemcitabine resistance.
8.2 Flow Cytometry Analysis
The aim of the flow cytometry analysis was to examine EGFR and HER2 expression was upregulated in GemR PANC-1 cells when compared to mock control, as outlined in the following sections.
Background:
The expression of EGFR and HER2 in two PANC-1 human pancreatic carcinoma cell lines (mock and gemcitabine resistant GemR) were by flow cytometry analysis.
Observation:
The expression of both EGFR and HER2 was upregulated in GemR PANC-1 when compared to their mock counterparts.
Conclusion:
Flow cytometry analysis showed that EGFR and HER2 were upregulated in the gemcitabine resistant pancreatic cancer cell line PANC-1. This result provides the rationale for targeting both EGFR and HER2 by Panobody to suppress cell growth and overcome gemcitabine resistance in pancreatic cancer cells
8.3 The effect of Panobody alone or in combination with gemcitabine in gemcitabine-resistant (GemR) PANC-1 cells.
Background:
(a) Gemcitabine resistant (GemR) PANC-1 was used to study the reversal activities of gemcitabine resistance by Panobody.
(b) Combined treatment of gemcitabine and Panobody is expected to demonstrate a synergistic effect and reverse gemcitabine resistance.
Observation:
(a) Panobody alone suppressed the cell proliferation of GemR PANC-1 cells.
(b) Panobody exerted a maximal synergistic growth suppressive effect in combination with gemcitabine in GemR PANC-1 cells.
The flow cytometry analysis confirmed our hypothesis that EGFR2 and HER2 are upregulated in our established gemcitabine resistant PANC-1 cells, which is confirmed by the MTT assay. The results of Bliss analysis demonstrated that our Panobody alone inhibited cell proliferation, while also exhibiting a synergistic growth-inhibiting effect when combined with gemcitabine. These promising initial findings suggest that Panobody represents a potential targeted therapy for pancreatic cancer, complementing gemcitabine treatment.
Conclusion:
Panobody demonstrated a synergistic effect in reversing the drug resistance of GemR PANC-1 cells to gemcitabine.