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
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 humanIL-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. Incubating the inoculum at 37℃ overnight.
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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