Engineering
Overview
The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits have become a severe healthcare problem[1]. Target therapies that turn off specific inflammation-causing genes have been proven as a major treatment in IBD[2]. In this project, a drug that interferes with Interleukin-18 is designed and tested to provide a new possible solution of IBD treatment IL-18 binding protein (IL-18BP) could bind to IL-18 to stop its function[3] (Fig 1). In humans, while there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), only IL-18BPa and IL-18BPc could antagonize IL-18 activity[4,5]. Plasmid containing IL-18 BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification is constructed and tested, the protein is then expressed and purified through BL21 E.coli strands. The purified protein was validated using Western Blotting and its activity was confirmed through T cell activation inhibition experiments.
Fig 1. The IL18 signal pathway diagram[3]
Cycle 1：BBa_K5522003 (pET28a-SUMO-IL-18BPa-Fc)
Design
The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPa, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPa-Fc fragment onto the plasmid using restriction digestion and ligation (Fig 2). We plan on testing this design within BL21 to verify its effectiveness in protein expression.
Build
The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPa genes fragments were also cut using the same enzymes and verified using gel electrophoresis.
The length of target genes SUMO-IL-18BPa-Fc is 1542bp. Fig 3A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 3.B shows that the plasmid was successfully digested.
The cut plasmid and the gene were ligated using T4 DNA ligase, and the recombinant plasmid was transformed into DH5alpha(Fig 4 A). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig. 4B).
Then 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 (Fig 5), indicating that the plasmid was successfully constructed.
The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPa, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPa-Fc fragment onto the plasmid using restriction digestion and ligation (Fig 2). We plan on testing this design within BL21 to verify its effectiveness in protein expression.
Fig 2. The plasmid map of pET28a-IL18-SUMO-BPa-Fc
The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPa genes fragments were also cut using the same enzymes and verified using gel electrophoresis.
The length of target genes SUMO-IL-18BPa-Fc is 1542bp. Fig 3A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 3.B shows that the plasmid was successfully digested.
Fig 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
B.Enzyme digestion product of pET-28α vector
Fig 4. The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPa .
A.pET-28α-Sumo-IL-BP-18-BPa-Fc-DH5α colony B. Verification of presence of Pa gene in DH5a transformants
A.pET-28α-Sumo-IL-BP-18-BPa-Fc-DH5α colony B. Verification of presence of Pa gene in DH5a transformants
Fig 5. Gene sequencing of IL-18-BPa.
Cycle 2：BBa_K5522002(pET28a-SUMO-IL-18BPc-Fc)
Design
The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPc, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPc-Fc fragment onto the plasmid using restriction digestion and ligation(Fig 6). We plan on testing this design within BL21 to verify its effectiveness in protein expression.
Build
The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPc genes fragments were also cut using the same enzymes and verified using gel electrophoresis.
The length of target genes SUMO-IL-18BPc-Fc is 1641bp. Fig 7 A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 7B shows that the plasmid was successfully digested.
The cut plasmid and the gene were ligated using T4 DNA ligase, and the recombinant plasmid was transformed into DH5alpha(Fig 8A). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig. 8B).
Then the plasmid pET-28a with Sumo-IL-18BP-Pc-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 (Fig 9), indicating that the plasmid was successfully constructed.
The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPc, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPc-Fc fragment onto the plasmid using restriction digestion and ligation(Fig 6). We plan on testing this design within BL21 to verify its effectiveness in protein expression.
Fig 6. The plasmid map of pET28a-IL18-SUMO-BPc-Fc
The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPc genes fragments were also cut using the same enzymes and verified using gel electrophoresis.
The length of target genes SUMO-IL-18BPc-Fc is 1641bp. Fig 7 A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 7B shows that the plasmid was successfully digested.
Fig 7. Identification of PCR amplified gene and enzyme digested pET-28α vector. A. PCR product of SUMO-IL-18BPc-Fc
B.Enzyme digestion product of pET-28α vector
B.Enzyme digestion product of pET-28α vector
Fig 8. The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPc .
A.pET-28α-Sumo-IL-BP-18-BPc-Fc-DH5α colony B. Verification of presence of Pc gene in DH5a transformants
A.pET-28α-Sumo-IL-BP-18-BPc-Fc-DH5α colony B. Verification of presence of Pc gene in DH5a transformants
Fig 9. Gene sequencing of IL-18-BPc.
Cycle 3: Test
3.1 SDS-PAGE
The purified IL18-BPa, IL18-BPc, and IL10-BP proteins were 56.9kDa, 61kDa, and 62.1kDa, respectively. The SDS-PAGE successfully verified the IL18-BPa, IL18-BPc, and IL10 proteins extracted and purified from E. coli BL21 (Fig 10).
3.2 Western Blot verification of extracted protein
Compared to coomassie Brilliant Blue staining, the principle of Western detection is antibody antigen specific reaction, with high detection specificity. The proteins we expressed all carrying His tag, and specific His antibodies can be used to detect purified proteins. As shown in Fig 11, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
3.3 Use BCA colorimetric method to determine the protein concentration
The concentration of the samples was measured using the BCA calorimetric method. We use standard concentration protein solution and BCA colorimetric method to determine the protein concentration. The absorbance value was measured by the enzyme-linked immunosorbent assay (ELISA) reader. We draw a scatter plot, insert a standard curve, and calculate the R-squared value.In the Fig.12, the fitting coefficient is greater than 0.99, indicating that the fitting effect is perfect.
The protein concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc were calculated according to the standard curve, as shown in Tab 3.
3.4.1 Measuring the standard curve using an INF-γ standard.
In the experiment, we need the concentration of INF-γ, and we establish the standard curve of INF-γ.The R squared value is larger than 0.99, which indicates that the curve is accurate (Fig. 13 ).
3.4.2 The influence of dose and storage conditions on protein activity
Store the three proteins (Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10) at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubate them with mouse primary T lymphocytes. Stimulate the cells with IL-18 and detect the IFN-γ in the cell supernatant.
In Fig 14, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ production.
The results are showed in Fig 14,the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 on IFN-γwas not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of Sumo-IL-BP-18-BPc, Sumo-IL-BP-18-BPa and Sumo-IL-10 protein on IFN-γ was significantly decreased. Storing at low-temperature preservation is beneficial for the stability of recombinant proteins. Furthermore, the anti-inflammatory effect of recombinant proteins is dose-dependent.
In the fig 15, it can be seen that after storage at 4°C for 24 hours, IL-18BPc's effects are still comparable to that of IL-10 in the 2.0 ug/ml, while IL-18BPa is less stable.
The purified IL18-BPa, IL18-BPc, and IL10-BP proteins were 56.9kDa, 61kDa, and 62.1kDa, respectively. The SDS-PAGE successfully verified the IL18-BPa, IL18-BPc, and IL10 proteins extracted and purified from E. coli BL21 (Fig 10).
Fig 10. SDS-PAGE verification of extracted proteins.
A, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc is red box, pET-28α-Sumo-IL-10 is blue box.
A, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc is red box, pET-28α-Sumo-IL-10 is blue box.
Compared to coomassie Brilliant Blue staining, the principle of Western detection is antibody antigen specific reaction, with high detection specificity. The proteins we expressed all carrying His tag, and specific His antibodies can be used to detect purified proteins. As shown in Fig 11, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
Fig 11. Detection of recombination protein expression by western blot. From left to right: pET-28α-Sumo-IL-10, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc. The size of Sumo-IL-18BPa-Fc is about 56.9 kDa; the size of Sumo-IL-18BPc-Fc is 61 kDa.the size of Sumo-IL-10-Fc is 62.1 kDa.
The concentration of the samples was measured using the BCA calorimetric method. We use standard concentration protein solution and BCA colorimetric method to determine the protein concentration. The absorbance value was measured by the enzyme-linked immunosorbent assay (ELISA) reader. We draw a scatter plot, insert a standard curve, and calculate the R-squared value.In the Fig.12, the fitting coefficient is greater than 0.99, indicating that the fitting effect is perfect.
Fig 12. Protein quantification using BCA colorimetric method.
Tab. 3 The concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc protein
3.4 Verification of Protein Function
| Sample | Concentration |
|---|---|
| Sumo-IL-18-BPa-Fc | 2.649213 μg/mL |
| Sumo-IL-18-BPc-Fc | 2.451683 μg/mL |
| Sumo-IL-10-Fc | 2.5152 μg/mL |
3.4.1 Measuring the standard curve using an INF-γ standard.
In the experiment, we need the concentration of INF-γ, and we establish the standard curve of INF-γ.The R squared value is larger than 0.99, which indicates that the curve is accurate (Fig. 13 ).
Fig 13. INF-γ quantification using ELISA.
Store the three proteins (Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10) at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubate them with mouse primary T lymphocytes. Stimulate the cells with IL-18 and detect the IFN-γ in the cell supernatant.
In Fig 14, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ production.
The results are showed in Fig 14,the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 on IFN-γwas not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of Sumo-IL-BP-18-BPc, Sumo-IL-BP-18-BPa and Sumo-IL-10 protein on IFN-γ was significantly decreased. Storing at low-temperature preservation is beneficial for the stability of recombinant proteins. Furthermore, the anti-inflammatory effect of recombinant proteins is dose-dependent.
Fig 14. The influence of dose and storage temperature on protein activity. Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc, and Sumo-IL-10A protein were stored at different temperatures, then were incubated with cells to determine the concentration of IFN γ in the cell supernatant.
Fig 15. The protein activity at 4°C. Ratio of IFN-gamma production levels after protein treatment vs control for IL-10 (B), IL-18BPa (C), IL-18BPc
Cycle 4: Learn
The testing in all three stages shows overall positive results, which indicates that our model design was reasonable and accurate. The result of the verifications also suggested that both IL-18BPa and IL-18BPc had similarly significant effect in anti-inflammatory therapies comparable to IL-10, hence, suggesting that these two proteins can serve as a basis for further experiments.
During the process of testing, we used an inefficient primer for the IL-18BPc gene, and redesigning the primers took some trial and error. We also realized two days within the experiment that we needed a positive control, that was why we bought a synthesized pET28a-SUMO-IL-10-Fc plasmid from GenScript and transformed it into BL21 cells along with our constructed plasmids. The end results demonstrated that SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-Fc had great potential to reduce inflammation, which give us future directions: for instance, our functionality tests occurred in vitro, but what would happen to the protein within the environment of the intestines, which is different in terms of pH, temperature, etc? What dose should be taken for different levels of inflammation? More questions are raised from the results, which is how research works.
During the process of testing, we used an inefficient primer for the IL-18BPc gene, and redesigning the primers took some trial and error. We also realized two days within the experiment that we needed a positive control, that was why we bought a synthesized pET28a-SUMO-IL-10-Fc plasmid from GenScript and transformed it into BL21 cells along with our constructed plasmids. The end results demonstrated that SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-Fc had great potential to reduce inflammation, which give us future directions: for instance, our functionality tests occurred in vitro, but what would happen to the protein within the environment of the intestines, which is different in terms of pH, temperature, etc? What dose should be taken for different levels of inflammation? More questions are raised from the results, which is how research works.
Reference
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https://doi.org/10.3389/fimmu.2022.919973