We applied the engineering cycle in the design and construction of BioBricks. The engineering cycle involves three main stages: 1) Design and Build, 2) Test, and 3) Learn. During the competition period, we went through three complete cycles, incorporating our observations, experimental results, literature review, suggestions and insights from each iteration. Utilizing this iterative process, we successfully proved a one-step cyclization and purification system using eGFP.
Phase 1: Design and Build
Bio-synthetic linear EGFR-binding peptide as the solution
Based on the results of the molecular docking simulation conducted in the dry lab, we identified a robust peptide sequence, CMYIEALDRYAF, which demonstrated a strong binding affinity towards EGFR. With this in mind, we chose this peptide sequence in linear form as the EGFR binding peptide (Ebp) and incorporated it into our first BioBrick design.
Phase 1: Test
To test our BioBrick, we synthesized the BioBrick and carried it by an expression vector, pET24d(+). The expression vector was transformed to TOP10 competent cells for plasmid amplification and to BL21(DE3) competent cells for expression. However, we encountered challenges during the peptide expression stage, potentially stemming from issues, including insolubility during protein extraction, peptide degradation or other unidentified factors, leading to an unsuccessful expression of the linear Ebp peptide. To resolve this matter, further investigation and troubleshooting are required to pinpoint the root cause and devise an appropriate solution.
Phase 1 : Learn
The failure of the peptide expression led to our concerns about the peptide degradation due to their short sequence length. To solve these challenges , we conducted literature review on the stability of linear peptides and sought guidance from professionals in the field. It had become evident that linear peptides were susceptible to proteolytic degradation and exhibited inherent instability. Exopeptidases tend to cleave terminal amino acids, leading to their rapid degradation. Moreover, without an optimized conformation, linear peptides often exhibit a relatively low target binding affinity and reduced potency. Additionally, linear peptide structures tend to have lower lipophilicity, resulting in a poor cell permeability. These findings highlighted the challenges associated with utilizing linear peptide and led us to consider alternative strategies, such as peptide cyclization or incorporating structural modifications, to enhance the peptide stability, binding affinity, and cellular permeability in peptide-based bispecific design.
Phase 2: Design and Build
One-step purification & cyclization system
After a thorough literature review and consultation with experts, we decided to pivot our design towards cyclic peptides. Acknowledging the unexpected outcomes resulted from phase 1, particularly the problem of the peptide stability and proteolytic degradation, we took a bold step to develop a system that integrates both peptide purification and cyclization in just one step.
Our one-step purification and cyclization system are based on four major components:
❖ Chitin Binding Domain (CBD)
The Chitin Binding Domain is a purification tag module that can be fused to other proteins to enable their purification using chitin-based affinity purification. N-terminus linked CBD and Its function is to bind specifically to chitin, allowing for the selective isolation of chitin-tagged proteins from a complex mixture of proteins.
❖ N-terminal: Ssp DnaB intein (Ssp)
The Ssp DnaB intein is a self-splicing protein domain that originated from the bacterium Synechocystis sp. PCC6803 and is capable of excising itself from its host protein (extein) through a series of self-catalysis reactions.
❖ C-terminal: modified Mxe GyrA intein (Mxe GyrA)
The Mxe GyrA intein is a self-splicing protein domain that originates from the bacterium Mycobacterium xenopi and is capable of excising itself from its host protein (extein) through a series of self-catalysis reactions.
❖ EGFR-binding peptide (Ebp)
The EGFR-binding peptide, CMYIEALDRYAF, is a 12 amino acid short peptide that binds with high affinity to the Epidermal Growth Factor Receptor (EGFR) as revealed by our docking simulation.
The chitin column plays a crucial role in achieving one-step purification and cyclization. The split inteins are flanked by chitin-binding domains (CBD) that enable the affinity purification of the BioBrick. This CBD exhibits a strong affinity for chitin beads, facilitating the efficient and straightforward recovery of the cyclic Ebp from the crude cell extract.
Phase 2: Test
We performed a series of molecular cloning and protein expression experiments, starting from the restriction digestion of the pET-24d(+) expression vector and the synthesized BioBrick DNA fragments to yield compatible sticky ends for subsequent ligation, to transformation and IPTG induced protein expression. We confirmed the success of the restriction digestion through DNA gel electrophoresis. The restriction digested expression vectors and gene fragments were then purified and subsequently subjected to ligation where it incorporated desired BioBricks to the pET-24d(+) expression vector. The recombinant plasmids were then heat-shock transformed to TOP10 competent cells for plasmid amplification and BL21(DE3) competent cells for protein expression. The expression was performed by inducing BL21(DE3) cells with IPTG at 25°C overnight. The proteins were extracted by the lysis buffer and subjected to SDS-PAGE analysis. In this phase, we successfully expressed the CBD-intein-Ebp-intein-CBD fusion protein. However, when attempting the protein purification using the chitin column, regrettably, no peptide was detected from the elution. The possible reason for this outcome may be attributed to the unsuitability of the Ssp-Mxe inteins for generating short peptides.
Phase 2: Learn
We confirmed that Ebp was successfully expressed as the CBD-intein-Ebp-intein-CBD fusion protein as shown in the SDS-PAGE analysis. Notably, the intein BioBrick housing Ebp did not undergo degradation during protein extraction and was soluble to the lysis buffer. This observation supported the idea that with a stronger structural complexity, Ebp tended to be more stable. Yet, the short Ebp still posed a challenge for this intein BioBrick, as it might not be suitable for cyclizing such a short peptide.
Phase 3: Design and Build
Upgrade of the Ssp-Mxe inteins to Npu inteins
We hypothesized that the purification failure was linked to the unsuitability of the Ssp-Mxe inteins for cyclizing short peptides. To address this, we retained the intein system while opting for another superior intein, the Npu intein. We learned from literature review that the Npus intein is capable of expressing peptides as short as six amino acids, which made it a promising solution for us to continue the wet-lab. To verify the design, we additionally added a eGFP positive for this BioBrick. In this phase, we involved three BioBricks: the target and scrambled peptide, and a eGFP positive control.
Phase 3: Test
The process of molecular cloning, transformation, protein expression and purification follow the same procedures as we had done in Phase 2. All groups including the Ebp, scrambled control and the eGFP controls were successfully expressed. By substituting the Ssp-Mxe inteins with the Npu inteins, we successfully obtained the cyclic eGFP from the Npu intein BioBricks. The calculated molecular mass of cyclic eGFP was 18 Da less than that of linear eGFP (26941 Da), resulting in a mass of 26923 Da, which indicated the linear eGFP has been modified into its cyclic form.
Phase 3: Learn
In Phase 3, we successfully completed the purification process of the eGFP control group and were ready for purify the Ebp and scrambled control. However, due to time constraints, we were unable to finish the purification process of Ebp and Scrambled control. It is worth noting that, the cyclic Ebp generated by the Npu intein system had not yet been confirmed during the iGEM competition period, but we achieved a success in purifying and cyclizing the linear eGFP to cyclic eGFP. This outcome validates the capability of the Npu intein system to purify and cyclize the short peptide.
Since we cannot further proceed to the next round of the engineering cycle, in order to show the efficacy and robustness of our novel bispecific antibody, Polyneerab, we used a chemically synthesized cyclic Ebp for antibody conjugation to serve as proof-of-concept purposes. The bispecific antibody was subjected to functional assays, such as MTT cytotoxicity assay and enzyme-linked immunosorbent assay (ELISA). Details of the experiment results can be found in the Experiment page.
Based on this encouraging preliminary data, Polyneerab is a promising agent to overcome Osimertinib-resistance in NSCLC.
