Part:BBa_K2817003
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.
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 is 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. Expressing vector harboring YebF and hIL-10 gene is transformed into BL21 strain. After induction of IPTG, we span down the cells, get them eliminated using filter. Secreted protein is 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 is demonstrated to show that more protein at 35kDA, which should be IL-10, is 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. Expressing vector is transformed into BL21 strain. CFU is then inoculated to LB medium followed by 12h incubation at 37℃. After induction of IPTG, final concentration at 1mM, the culture is incubated at 37℃ for 2h. A coomassie brilliant blue is 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 add 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. Expressing vector is transformed into BL21 strain. After induction of nitric oxide, the culture is incubated at 37℃ for 2 hours. Western blotting is demonstrated to show the expression level of hIL-10. In order to equate the total number of cells in each lane, dilution is made to keep each sample at the same OD and 10μL of each sample is uploaded to each lane.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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