(fig a. our recombinant plasmid design, utilizing the pUC18 vector.)
Our objective is to biosynthesize the fusion protein TP1_P5 in E.coli under optimal culture conditions. To achieve this, we have designed a recombinant plasmid specifically tailored to facilitate the expression of our target protein.
The plasmid incorporates a T7 promoter, which ensures robust transcription of our insertion when induced. Following the promoter, we included a ribosome binding site (RBS) to enhance translation efficiency. This design features multiple start codons, allowing flexible initiation of translation based on the cellular environment.
The fusion protein sequence is strategically positioned downstream of the RBS, followed by a TEV cleavage site for the post-expression processing of our fusion protein, enabling efficient cleavage to release TP1_P5 from any fusion partners or tags. Additionally, we have incorporated a 6x histidine tag (His-tag) to facilitate the purification process via metal affinity chromatography, which allows for specific binding to nickel ions during purification.
The construct ends with stop codons and a double terminator to ensure proper termination of transcription.
We designed our gene construct based on the pUC-18 vector, and we inserted our recombinant plasmid into two types of E. coli. , DH5 Alpha and BL21, and we purified the proteins to prove that TP1P5 is present.
The synthetic gene block containing our genes of interest (TP1 and P5) was transformed into DH5 Alpha and BL21. The two types of cells were then frozen and prepared for cell culture.
50μL of the cell samples were spread on agar gel and incubated at 37°C. A colony of the cells was then selected and used for cell culture. Thus, we have a sufficient amount of cells containing the plasmid containing our fusion protein.
The cells were then underwent protein purification to obtain a pure sample of our protein to confirm the presence of TP1P5 without contamination and interference from other proteins.
We conducted gel electrophoresis alongside the SDS-PAGE method to prove the existence of our proteins. These methods could provide us the length of both the DNA and the protein. By comparing the position of the protein of interest to the protein ladder, we could deduce the size of the protein. If the position of our protein relative to the protein ladder corresponds to the theoretical size of our plasmid, then the experiment would be deemed successful.
We believe that the expression of the protein in the bacteria is insufficient, according to the faint colour of our protein band in the gel demonstrated. Henceforth, we have to navigate a method that can increase the production of our fusion protein inside E. coli, such as incorporating another protein strain inside of our plasmid that can increase the synthesis of our gene of interest.
We subsequently developed new versions of the recombinant plasmid that incorporate the DSBA domain. DSBA is a disulfide bond isomerase that catalyses intrachain disulfide bond formation, which is essential for the proper folding of proteins that contain disulfide bonds. By integrating the DSBA sequence into our plasmid. We aim to enhance the expression and stability of our target fusion protein, TP1_P5, in the E. coli BL21 strain, and for that, we have switched to another transcription vector, pET-21, designed for expression from bacterial translation signals carried within a cloned insert.
(fig b. the separately expressed DSBA plasmid, using the pET-21 vector)
Aside from that, we have also considered the varying severity and scenarios of the cardiovascular diseases and to prevent overdosing, we decided to express TP1 and P5 separately. Similarly, we have incorporated a 6x His-Tag to facilitate the purification process as round one.
Ginsentide TP1 demonstrates a multifaceted approach to promoting cardiovascular health through its activation of the PI3K-Akt pathway and subsequent effects on nitric oxide production, vascular relaxation, and platelet aggregation inhibition.
Lupin peptide P5 has emerged as a promising candidate in cholesterol management, exhibiting statin-like properties through its competitive inhibition of HMG-CoA reductase. By targeting this crucial enzyme in the mevalonate pathway, Lupin peptide P5 plays a significant role in reducing cholesterol production and enhancing the uptake of low-density lipoprotein (LDL) particles.
When our fusion protein is in action, it will accumulate in certain regions of the afflicted blood vessel. The concentration of our in-action fusion protein would vary depending on the severity of the damage of the blood vessel. The more concentrated our proteins are, indicating the more severe the infection is. This is where our newly implemented green fluorescent protein (GFP), and a smaller luminescent probe (miRFP670nano) comes into play, where it acts as an alternative diagnostic tool, indicating the locations of our fusion proteins, and we can subsequently tell the concentration of our fusion protein, alongside the severity of the situation. This can demonstrate the current situation of the blood vessel, and professionals could judge and decide whether immediate surgery should be carried out, while preventing the situation from worsening.
Through the regulation of tp1 and p5 dosages in our product, we can offer personalised medicine for patients with different extents of endothelial injury platelet aggregation and LDL level; and with the addition of GFP and the probe, we could provide a more affordable alternative of diagnostic measures for both professionals and laymen.
(fig c. Our revised recombinant plasmid featuring GFP, utilizing the pET-21 vector)
(fig d. Our revised recombinant plasmid featuring miRFPnano, utilizing the pET-21 vector)
To maximise the effectiveness of this approach, we will first transform the BL21 strain with the plasmid containing the DSBA part. This initial transformation will enable the bacteria to express DSBA, increasing the availability of this important chaperone protein within the cells. The presence of DSBA is expected to facilitate the proper folding of our target protein, thereby improving the yield of correctly folded TP1_P5.
After establishing a stable BL21 strain with DSBA expression, we will proceed with a second round of transformation to introduce our recombinant plasmid containing TP1_P5. This two-step transformation strategy not only ensures a higher concentration of DSBA in the bacterial system but also allows us to create a more favourable environment for the expression of disulfide-bonded proteins.