pET28a-IL18-BPa

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 4402
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370
Illegal NgoMIV site found at 5597
1000
COMPATIBLE WITH RFC[1000]

SUMO-IL18-BPa-Fc: Construction and Design

Construction Design

The modified human BPa gene was designed as described previously in the "Add a new basic part" section. The pET-28alpha containing kanamycin resistance and another His tag at the N-terminal was obtained from GenScript. The plasmid and gene fragment were cut using restriction enzymes NheI and XhoI, then recombined using ligation enzymes to form the recombinant plasmid (Figure 1).

Figure 1. The plasmid map of pET28a-IL18-SUMO-BPa-Fc

Engineering Principle

The spread of Inflammatory Bowel Disease (IBD) in Asian countries, due to the adoption of Western dietary habits, has become a severe healthcare problem in recent years¹. Targeted therapies that turn off specific inflammation-causing genes have been proven to be a major treatment for IBD². In this project, a drug that interferes with Interleukin-18 (IL-18) is designed and tested to provide a potential solution for IBD treatment. IL-18 binding protein (IL-18BP) binds to IL-18 to stop its function³ (Figure 2). In humans, there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), and IL-18BPa is known to antagonize IL-18 activity^4,5. A plasmid containing IL-18BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification was constructed and tested. The protein was expressed and purified through BL21 E. coli strains. The purified protein was validated using Western Blotting, and its activity was confirmed through T-cell activation inhibition experiments.

Figure 2. The IL-18 signal pathway diagram

Experimental Approach

The PET-28α blank plasmid was cut using restriction enzymes to make it linear. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. After the DNA was recovered from the gel, the concentration and purity of the samples were measured. The length of the target gene SUMO-IL-18BPa-Fc is 1542bp. Figure 3A shows that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Figure 3B shows the plasmid was successfully digested.

Figure 3. Identification of PCR amplified gene and enzyme digested pET-28α vector. A. PCR product of SUMO-IL-18BPa-Fc. B. Enzyme digestion product of pET-28α vector.

After restriction enzyme digestion and ligation of pET-28a with Sumo-IL-18BP-Pa-Fc, the recombinant plasmids were transformed into E. coli DH5α competent cells (Figure 4A). The transformants were identified by colony PCR, and the agarose gel electrophoresis results showed the expected length of PCR products, confirming successful construction (Figure 4B).

Figure 4. A. pET-28α-Sumo-IL-BP-18-BPa-Fc-DH5α colony. B. Verification of presence of Pa gene in DH5α transformants.

The plasmid pET-28a with Sumo-IL-18BP-Pa-Fc was sent to the biological company for sequencing. The comparison of the sequencing results showed that the target gene sequence was consistent with the sequencing results, indicating that the plasmid was successfully constructed (Figure 5).

Figure 5. Gene sequencing of IL-18-BPa.

Cultivation, Purification and SDS-PAGE

The purified IL18-BPa protein was 56.9 kDa. SDS-PAGE successfully verified the IL18-BPa proteins extracted and purified from E. coli BL21 (Figure 6).

Figure 6. SDS-PAGE verification of extracted proteins.

Compared to Coomassie Brilliant Blue staining, Western detection's principle involves antibody-antigen specific reactions, providing high detection specificity. The proteins we expressed all carried a His tag, and specific His antibodies were used to detect purified proteins. As shown in Figure 7, the protein size we obtained was consistent with the expected size, demonstrating successful protein expression.

Western blot detection of recombination protein expression

Figure 7. Detection of recombination protein expression by western blot. From left to right: pET-28α-Sumo-IL-10, pET-28α-Sumo-IL-BP-18-BPa, pET-28α-Sumo-IL-BP-18-BPc. The size of Sumo-IL-18BPa-Fc is about 56.9 kDa.

Characterization/Measurement

The activity of the protein was characterized by its ability to inhibit T-cell activation. Mice abdominal T-cells were stimulated by TNFalpha and IL-18. Recombinant protein was added, and the production of IFN-gamma was measured using ELISA. The results showed that the recombinant protein was able to inhibit the production of inflammation-inducing IFN-gamma, indicating its anti-inflammatory effect (Figure 8).

Figure 8. The influence of storage temperature on protein activity.

References

[1] Piersiala K, Hjalmarsson E, da Silva PFN, Lagebro V, Kolev A, Starkhammar M, Elliot A, Marklund L, Munck-Wikland E, Margolin G, Georén SK, Cardell LO. Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes. Int J Cancer. 2023 Aug 15;153(4):854-866. doi: 10.1002/ijc.34555. Epub 2023 May 5. PMID: 37144812.
[2] Liang X, Fan Y. Bidirectional two-sample Mendelian randomization analysis reveals a causal effect of interleukin-18 levels on postherpetic neuralgia risk. Front Immunol. 2023 May 25;14:1183378. doi: 10.3389/fimmu.2023.1183378. PMID: 37304287; PMCID: PMC10247971.
[3] Menachem A, Alteber Z, Cojocaru G, et al. Unleashing Natural IL-18 Activity Using an Anti-IL-18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects. Cancer Immunology Research. 2024. OOF1-OF17.
[4] Yamanishi K, Hata M, Gamachi N, et al. Molecular Mechanisms of IL-18 in Disease. International Journal of Molecular Sciences. 2023, 24(24).
[5] Ihim, S. A., Abubakar, S. D., Zian, Z., Sasaki, T., Saffarioun, M., Maleknia, S., & Azizi, G. Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment. Frontiers in Immunology. 2022. 13: 919973. https://doi.org/10.3389/fimmu.2022.919973