constitutive expression of CBDcex fused to INPNC with 6xHis tag
CBDcex fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be the therapeutic solution against Norovirus infection. This protein in particular is a cellulose binding domain(CBDcex) fused to surface expression anchoring domain(INPNC), connected by a 6xHis tag to be easily identified by Western blotting.
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Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21Illegal BamHI site found at 1012
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 132
Illegal NgoMIV site found at 465
Illegal AgeI site found at 883
- 1000COMPATIBLE WITH RFC
We succeeded in surface expressing anti-NoV scFv BBa_K1933002 antibody using INPNC-His. We aimed to orally administrate this protein expressing E. coli, however, we obtained an overwhelmingly negative result from surveys conducted when asked "Would you feel uncomfortable with oral administration of sterilized recombinant organisms?" This is why we also constructed a parts that surface expresses cellulose binding domains (CBD). With this part, we can "leash" E.coli to cellulose and make them swiftly exit human digestive systems. We selected CBDcex as a CBD for this part.
This part had an important role in integrating our Human Practice activities into our project.
We designed PCR primers so that we would obtain PCR product with the length of 311 bp after RT-PCR. For negative control, reverse transcription was not performed.
A band corresponding to 311 bp was observed only in INP-His-scFv sample, not in negative control(Fig.1). This result suggests that INP-His-CBDcex was successfully transcribed.
We used whole-cell Western blotting with anti-His tag antibody(Fig.2(a)). Then, we prepared the membrane fraction from the E. coli lysate. Membrane fraction was solubilized and used for Nickel Sepharose purification and precipitates were examined by Western blotting against His tag(Fig.2(b)).
A band corresponding to INPNC-His-CBDcex (47kDa) was observed in both whole cell and membrane fraction Western blotting (Fig.2(a),(b)), which confirms expression of the fusion proteins, and suggests that CBDcex was surface expressed, respectively.
As shown in Fig3, we observed the expression of INPNC-His-CBDcex on the membrane fraction. We next tested CBDcex's binding to cellulose.
Fig3 shows the results. After incubating E. coli with about 35~38 micrometer cellulose powder, we DAPI stained E. coli and observed it under fluorescence microscopy. As shown, CBD-surface expressing E. coli was observed binding to cellulose, showing the functional expression of INPNC-His-CBD in our system. When control plasmid was used, the numbers of cellulose-bound E. coli cells were significantly reduced (Fig4).
From these results, we concluded that this part can be used to physically link E. coli and cellulose.
We fulfilled criteria listed below with this part.