Part:BBa_K4766013
DogCatcher-Mi3
We linked DogCatcher to Mi3 so that it can capture proteins connected with DogTag.
In our project, we aim to determine whether the lab-engineered protein cage Mi3 can serve as a universal detection tool, applicable for rapid diagnostics in various areas such as disease biomarkers, inflammatory factors, or environmental pollutants. Once the Mi3 subunit is fused with a coupling tag, it can assemble with cargo proteins that also possess this coupling tag. During expression, 60 Mi3 subunits can spontaneously assemble to form a dodecahedral protein cage called Mi3. By utilizing the 60 sites on the protein cage, we introduce a high ratio of eGFP fluorescent reporter molecules to illuminate the protein cage. Additionally, by introducing a specific ratio of particular antibody molecules and leveraging immunorecognition, we aim to capture biomarkers, facilitating the efficient detection of specific biological molecules.
Having obtained the amino acid sequences of Mi3 and DogCatcher, we engineered the recombinant plasmid pET28a-DogCatcher-Mi3 using the pET28a vector, depicted in Figure 1. We then used PCR amplification to verify the presence of the target fragment in pET28a-DogCatcher-Mi3 that includes DogCatcher-Mi3, as illustrated in Figure 2.
Fig.1 Schematic design of the target protein gene fragment for the recombinant plasmid pET28a-DogCatcher-Mi3.
Fig.2 Electrophoresis image of the PCR fragment of recombinant plasmid pET28a-DogCatcher-Mi3.
Following our initial steps, we tried expressing the SnoopCatcher-Mi3 protein from our recombinant plasmid, depicted in Figure 3. Yet, the overwhelming formation of inclusion bodies by SnoopCatcher-Mi3 greatly complicated protein purification. As a result, we opted out of using the DogCatcher/DogTag pairing for our project's biological coupling system.
Fig.3 Solubility analysis of DogCatcher-Mi3. Lane 1: Resuspended pellet of lysed E.coli BL21 (DE3) Lane 2: Supernatant of lysed E.coli BL21 (DE3).
Sequence and Features| None |