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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.
2.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.
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.
2.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 1A, 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 1B).
The purified IL10-BP protein was 62.1kDa. The SDS-PAGE successfully verified the IL10 proteins extracted and purified from E. coli BL21 (Fig 2).
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 3, 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 4, 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 4 , 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.
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 1A, 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 1B).
Fig 1. Gene sequencing and colony PCR amplification of sumo-IL10-Fc
Fig 2. SDS-PAGE verification of extracted proteins.
Fig 3. Detection of recombination protein expression by western blot.
In Fig 4, 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 4 , 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.
Fig 4. The influence of dose and storage temperature on protein activity.Sumo-IL-10 protein was stored at different temperatures, then were incubated with cells to determine the concentration of IFN γ in the cell supernatant.
Reference
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[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.