In our project, we have added some new parts to iGEM part (Table 1). This includes a newly synthesized gene IL18-BPa( BBa_K5522001 ), IL18-BPc( BBa_K5522000 ) and IL10.And there expression vectors: pET28a-IL18-SUMO-BPa-Fc ( BBa_K5522003 ), pET28a-IL18-SUMO-BPc-Fc ( BBa_K5522002 ) and pET28a-IL10 ( BBa_K5522005 , positive contrast). This project designed two recombinant IL-18 binding proteins. Their structure includes natural IL-18 binding proteins, IgG1 antibody Fc fragments, and SUMO for protein stability.

1.Add New Basic Parts
1.1 BBa_K5522001 (IL18-BPa)
Name: SUMO-IL18-BPa-Fc
Base Pairs: 1542bp
Origin: Homo sapiens
Properties: The protein encoded by this gene is a proinflammatory cytokine of the IL-1 family that is constitutively found as a precursor within the cytoplasm of a variety of cells including macrophages and keratinocytes. The inactive IL-18 precursor is processed to its active form by caspase-1, and is capable of stimulating interferon gamma production, and of regulating both T helper (Th) 1 and Th2 responses. This cytokine has been implicated in the injury of different organs, and in potentially fatal conditions characterized by a cytokine storm. In humans, IL-18 gene is located on chromosome 11. Alternatively spliced transcript variants encoding different isoforms have been found for this gene1-2.
Usage and Biology:
IL18 has been used in many fields. IL18BP is induced in the tumor microenvironment (TME ) in response to the upregulation of IFNγ in the negative feedback mechanism for the development of cancer drugs3. And such as alzheimer 's disease have been studied4. On this basis, we used to IL18 treat IBD. The SUMO modification could enhance the stability of the protein. Among them, human IL-18BP has four different subtypes of IL-18BPa, IL-18BPb, IL-18BPc and IL-18BPd, of which only IL-18BPa has the biological function of inhibiting IL-18, and is an ideal candidate drug for blocking IL-18 treatment of IBD4-5 .In the our project, the Fc modification was shown by other research to slow the degradation of proteins in vivo. The His tag was used for protein purification. Codon optimization improves gene expression and enhance translational efficiency within E. coli.
1.2 BBa_K5522000 (IL18-BPc)
Name: SUMO-IL18-BPc-Fc
Base Pairs: 1641bp
Origin: Homo sapiens
Properties:The protein encoded by this gene is a proinflammatory cytokine of the IL-1 family that is constitutively found as a precursor within the cytoplasm of a variety of cells including macrophages and keratinocytes. The inactive IL-18 precursor is processed to its active form by caspase-1, and is capable of stimulating interferon gamma production, and of regulating both T helper (Th) 1 and Th2 responses. This cytokine has been implicated in the injury of different organs, and in potentially fatal conditions characterized by a cytokine storm. In humans, IL-18 gene is located on chromosome 11. Alternatively spliced transcript variants encoding different isoforms have been found for this gene1-2.
Usage and Biology:
There are four different subtypes of human IL-18BP, IL-18BPa, IL-18BPb, IL-18BPc and IL-18BPd. IL-18BPc can inhibit the biological function of IL-18, and human IL-18BPa shows the greatest affinity to IL-18, which is an ideal candidate drug for blocking IL-18 in the treatment of IBD3-5.In our project, the DNA sequence of human IL-18BPc gene was obtained from research article Novick et al. The sequences coding for SUMO and IgG1 Fc were both obtained from NCBI Homo sapiens genome. The sequences were compiled in this fashion: SUMO-IL-18BPc-Fc. 6×His tag was added to C terminal of the protein. The SUMO modification could enhance the stability of the protein. The Fc modification was shown by other research to slow the degradation of proteins in vivo. The His tag was used for protein purification. Codon optimization improves gene expression and enhance translational efficiency within E. coli.
2.New composite parts
2.1 BBa_K5522003 (pET28a-IL18-SUMO-BPa-Fc) 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 pasmid and gene fragment were cut using restriction enzymes NheI and XhoI, then recombined using ligation enzymes to form the recombinant plasmid (Fig 1).
Engineering Principle:
The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits have become a severe healthcare problem in recent years1. Target therapies that turn off specific inflammation-causing genes have been proven as a major treatment in IBD2. 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 function3 (Fig 2). 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 activity4,5. 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.
Experimental Approach
The PET-28α blank plasmid was cut by restriction enzymes to make them 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 gel, the concentration and purity of the samples were then measured.
The length of target genes SUMO-IL-18BPa-Fc is 1542bp. Figure 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. Figure 3.B shows that the plasmid was successfully digested.
After restriction enzyme digest and ligation of pET-28a with Sumo-IL-18BP-Pa-Fc, the recombinant plasmids were transformed into E.coli DH5α competent (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.
Cultivation, Purification and SDS-PAGE
The purified IL18-BPa proteins was 56.9kDa. The SDS-PAGE successfully verified the IL18-BPa proteins extracted and purified from E. coli BL21 (Fig 6).
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 figure 7, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
Characterization and Measurement
The activity of protein was characterized by its ability to inhibit T cell activation. Mice abdominal T-cells were stimulated by TNFalpha and IL18, recombination protein was added and the production of IFN-gamma measured by ELISA. The result showed that, the recombination protein was able to inhibit the production of the inflammation-inducing IFN-gamma, indicating its anti-inflammatory effect(Fig 8).
2.2 BBa_K5522002 (pET28alpha-IL18-SUMO-BPc-Fc)
Construction Design
The modified human BPc 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. pasmid and gene fragment were cut using restriction enzymes NheI and XhoI, then recombined using ligation enzymes to form the recombinant plasmid (Fig 9).
Experimental Approach;
The PET-28α blank plasmid was cut by restriction enzymes to make them 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 gel, the concentration and purity of the samples were then measured.
The length of target genes SUMO-IL-18BPc-Fc is 1641bp. Figure 10A 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. Figure 10.B shows that the plasmid was successfully digested.
After restriction enzyme digest and ligation of pET-28a with Sumo-IL-18BP-Pc-Fc, the recombinant plasmids were transformed into E.coli DH5α competent (Fig 11A). 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. 11 B).
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 12 ), indicating that the plasmid was successfully constructed.
Cultivation, Purification and SDS-PAGE
The purified IL18-BPc proteins was 61kDa. The SDS-PAGE successfully verified the IL18-BPa proteins extracted and purified from E. coli BL21 (Fig 13).
2.3 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 figure 14, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
Characterization and Measurement
The activity of protein was characterized by its ability to inhibit T cell activation. Mice abdominal T-cells were stimulated by TNFalpha and IL18-BPc, recombination protein was added and the production of IFN-gamma measured by ELISA. The result showed that, the recombination protein was able to inhibit the production of the inflammation-inducing IFN-gamma, indicating its anti-inflammatory effect(Fig 15 ).
2.3 BBa_K5522005 (pET28a-IL10)
Summary:
In order to provide protein expression and solubility, we design to add different tags and codon optimization.
1.The sequences were compiled in this fashion: SUMO - IL-10 - Fc( BBa_K5522004 ). 6×His tag was added to C terminal of the protein. The SUMO modification could enhance the stability of the protein. The Fc modification was shown by other research to slow the degradation of proteins in vivo. The His tag was used for protein purification.
The codon optimization improves gene expression and enhance translational efficiency within E. coli. And we constructed a new plasmid BBa_K5522005 (pET28a-IL10), and induced protein expressionand tested inhibition IFN-gamma, indicating its anti-inflammatory effect. Documentation
a.Usage and Biology: The protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. This cytokine has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines, MHC class II Ags, and costimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. This cytokine can block NF-kappa B activity, and is involved in the regulation of the JAK-STAT signaling pathway. Knockout studies in mice suggested the function of this cytokine as an essential immunoregulator in the intestinal tract. Mutations in this gene are associated with an increased susceptibility to HIV-1 infection and rheumatoid arthritis1.
b.Characterization/Measurement
We transformed the pET28a-Sumo-IL10-Fc synthesized by the company into E.coli BL21 to promote protein expression as our subsequent positive control. In the fig 16 A, the agarose gel electrophoresis results showed that we obtained the expected length of sumo-IL10-Fc, indicating the construction were successfully completed. And the target gene sequence was consistent with the sequencing results(Fig 16B).
The purified IL10-BP protein was 62.1kDa. The SDS-PAGE successfully verified the IL10 proteins extracted and purified from E. coli BL21 (Fig 17).
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 18, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
Store the the proteins 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 19, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-10 proteins on IFN-γ production.
The results are showed in fig 19 , the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-10 on IFN-γ was not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of 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-dependen.