Difference between revisions of "Part:BBa K2817003"

Latest revision as of 06:51, 29 September 2024

YebF-IL10-flag

IL10 is a natural immunosuppressive protein. We add a flag tag for detection and a secretion tag to increase the extracellular concentration.

Improvement made by 2024 SHSID-China

Uploaded by Hayashi Rika

Summary

In order to increase protein expression and solubility, we designed different tags and applied codon optimization techniques.

1. The sequences were compiled in this fashion: SUMO - IL-10 - Fc (BBa_K5522004). A 6×His tag was added to the C-terminal of the protein. The SUMO modification enhances the stability of the protein. The Fc modification slows the degradation of proteins in vivo. The His tag is used for protein purification.
2. Codon optimization improves gene expression and enhances translational efficiency within E. coli. We constructed a new plasmid BBa_K5522005 (pET28a-IL10), induced protein expression, and tested inhibition of IFN-gamma, indicating its anti-inflammatory effect. At the same time, we compared our new part with IL18.
3. Our project provides a potential new treatment for IBD and compares the effects of several different proteins.

Documentation

Usage and Biology

The protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. It has pleiotropic effects in immunoregulation and inflammation, down-regulating 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 suggest its role as an essential immunoregulator in the intestinal tract. Mutations in this gene are associated with an increased susceptibility to HIV-1 infection and rheumatoid arthritis 1.

The pET28a vector is widely used in synthetic biology, containing a T7 promoter and lac operator, allowing for fine-tuned control over the plasmid's recombinant protein expression (induced by IPTG). It includes a poly-histidine sequence for easier purification, and kanamycin resistance for selecting bacterial colonies. After combining the human SUMO and Fc gene fragments (from NCBI), we applied codon optimization and added NheI and XhoI restriction sites for ligation (Fig 1). We plan to test this design in BL21 to verify its effectiveness in protein expression.

Characterization/Measurement

We transformed the pET28a-Sumo-IL10-Fc synthesized by the company into E. coli BL21 to promote protein expression as our positive control. In Fig 2A, agarose gel electrophoresis results showed that we obtained the expected length of sumo-IL10-Fc, indicating successful construction. The target gene sequence was consistent with the sequencing results (Fig 2B).

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.

2019 NEU-CHINA

Characterization result 1

Secretory tag YebF was introduced to secret the protein, however we did not have time to show this tag really work last year. This time, western blotting was demonstrated to show the secretion of human IL-10. Expressing vector was constructed.

Figure 1. Diagram for  human IL-10 expressing and secreting in pCDFDuet-1 plasmid. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. LacO, the sequence represses the nearby promoter when there is no inducer (e.g. IPTG). RBS, ribosome biding site. Secretory tag YebF is introduced to secret protein downstream.

Figure 2. Protein expression of human IL-10 gene which transformed in BL21 strain. After induction of IPTG, we span down the cells, get them eliminated using filter. Secreted protein was collected from the medium by salting out with ammonium sulfate. The molecular weight of protein YebF-hIL-10 is around 35 kDa.

Protocol

1. Incubate the inoculum at 37℃ overnight.

2. Dilute the culture to OD600=0.2, followed by incubation at 37℃ for around 2 hours until the OD600=0.6.

3. Add IPTG at final concentration 1mM.

4. Incubate the culture overnight.

5. Remove cells by centrifugation at 5000rpm for 10min.

6. Remove cells by using filter.

7. Add saturated ammonia sulfate to the supernatant, and then agitate softly for 2 hours while sitting on ice.

8. Span the protein down by centrifugation at 13000rpm for 30min.

9. Resuspend the protein by cold water and remove any faults by filter.

10. Add Loading Buffer and then incubate the mixture at 98℃ for at least 10min.

11. Western blot.

Characterization result 2

To make sure that amplifier is able to strengthen the expression of gene downstream to promoter hrpL. A coomassie brilliant blue was demonstrated to show that more protein at 35kDA, which should be IL-10, was witnessed when introducing IL-10 gene downstream of promoter hrpL.

Figure 3. Diagram of expressing vector. HrpR and HrpS amplifier system is used as a booster to boost the expression level of gene downstream promoter hrpL. Protein HrpR and HrpS can bind together to induce the PhrpL which strengthen the expression of downstream gene YebF-IL10.

Figure 4. Protein expressing of human IL-10 gene which was transformed into BL21 strain. CFU was then inoculated to LB medium followed by 12h incubation at 37℃. After induction of IPTG, final concentration at 1mM, the culture was incubated at 37℃ for 2h. A coomassie brilliant blue was demonstrated to show the result. The red box shows where the target protein is.

Characterization result 3

In order to see whether the nitric oxide sensor will work when it comes to expressing a certain protein, we added the IL-10 downstream and demonstrated the western blotting to show the result. It has been proved that the expression level can be induced when adding nitric oxide.

Figure 5. Diagram of expressing vector in backbone pCDFDuet-1. Promoter yeaR is an nitric oxide inducible promoter. Gene hIL-10 is one of our target protein.

Figure 6. Protein expressing of human il-10 gene which was transformed into BL21 strain and induced by nitric oxide. After induction of nitric oxide, the culture was incubated at 37℃ for 2 hours. Western blotting was demonstrated to show the expression level of hIL-10. In order to equate the total number of cells in each lane, dilution was made to keep each sample at the same OD₆₀₀ and 10μL of each sample was uploaded to each lane.

Characterization result 4

It is very important to know the bio-activity and expressing efficiency of IL-10 expressed by our bacteria strain, since we are targeting the medical use of IL-10. We use human IL-10 ELISA kit (BOSTER Biological Technology co.Itd) to do the activity assay. It is notable that all our samples showed great IL-10 bio-activity, and the expressing level was super high. After incubation at 16℃ for 20h, the concentration of IL-10 in the culture can be 23 ng/ml.

Figure 7. Result of activity assay using human IL-10 ELISA kit. Wells C1-C8 are atandard human IL-10 protein sample with concentration at 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml, 7.8pg/ml and 0pg/ml respectively. Wells B1-B4 are serial dilution of whole protein sample 1 with 1x, 10x, 100x and 1000x, while B5-B8 are serial dilution of whole protein sample 2. Wells A1-A3 are serial dilution of secret protein sample 1 with 1x, 10x and 100x while wells A4-A6 are serial dilution of secret protein sample 2. Well A7 and A8 are control. Sample 1 and sample 2 are amplified cells from different CFUs after transforming expressing vector into BL21 strain.

Figure 8. Standard curve of human IL-10.

Figure 9. Result of activity assay using human IL-10 ELISA kit. CFUs were inoculated to LB medium followed by 12h incubation at 37℃. After induction of IPTG, the culture was incubated at 16℃ for 20h. Cells and medium were collected and ready for assay. The volume of culture for whole protein was 10ml while the volume of culture for secret protein is 50ml.

Sequence and Features