Part:BBa_K5293006:Experience
Applications of BBa_K5293006
This insert has been tested in multiple different vectors and validated through several different experiments. Early in our project, we cloned it into pCLGGx vectors with subcellular localization tags encoding recombinant protein expression at those particular locations. These were transformed into Agrobacterium tumefaciens and infiltrated in the leaves of plant Nicotiana benthamiana, which were collected and analysed for mScarlet-semaglutide expression.
The His-tag adds great value to this part, as it enables immunoblot analysis. Conducting Western blots with anti-His antibody allows for specific identification of mScarlet-semaglutide among all other proteins in the crude lysate. Below is a figure of His-mScarlet-TEV-Sema, expressed at ~33 kDa and identified by anti-His binding.
Figure 1. Comparative subcellular localizations of pCLGGX-His-mScarlet-TEV-Semaglutide heterologously expressed in Nicotiana benthamiana at two different levels of bacterial infection (Optical density of 0.25 and 0.5). All constructs were co-infiltrated with the pHREAC backbone for its suppressor of silencing feature. 12%, 1.5mm Tris Glycine SDS-PAGE gels were used to conduct Western Blot analyses (bottom right), accompanied by a Coomassie brilliant blue staining as loading control (top right). The estimated concentrations, based on a Bradford assay, are given in the table (bottom left). An image of the leaves at the time of harvest are also provided.
The expression of His-mScarlet-TEV-Sema has also been validated by conducting a plate reader assay screening for mScarlet fluorescence. Comparison to a purified mScarlet positive control allowed for quantification of the amount of mScarlet present after infiltrations
Figure 2. Plate reading assay results measuring mScarlet fluorescence for comparative subcellular localizations of pCLGGX-His-mScarlet-TEV-Semaglutide heterologously expressed in Nicotiana benthamiana. Assay is conducted using an excitation wavelength of 564 nm, emission wavelength of 610 nm, and bandwidth of 20 nm. Quantification of the amount of mScarlet present in each sample done via comparison to a standard curve of purified mScarlet.
Expression of His-mScarlet-TEV-semaglutide has also been achieved by cloning it into the pHREAC vector (used for effective biopharming). Western blot analysis also shows high levels of expression of this protein by validating the strong presence of a His-tagged protein at ~33 kDa.
Another significant value of this part is its applicability to protein purification. The presence of a His tag allows for binding to a His column during fast protein liquid chromatography. Analysis of purified mScarlet-semaglutide by affinity chromatography is shown below:
Figure 3. SDS-PAGE results of affinity chromatography fractions from pHREAC-His-mScarlet-TEV-Semaglutide heterologously expressed in Nicotiana benthamiana. Fast protein liquid chromatography used (AKTA Pure) with HisTrap HP 1 ml column (Cytiva). 12%, 1.5mm Tris Glycine SDS-PAGE gels were used to conduct Coomassie brilliant blue staining (left) and Western Blot analyses (right).
His-mScarlet-semaglutide is clearly visible at ~33 kDa, demonstrating how the His-tag allows for purification among an input of multiple contaminant proteins.
Furthermore, the TEV site allows for specific cleavage between mScarlet and semaglutide by incubation with a TEV protease. His tag affinity purification allows for the separation of these two parts of the fusion protein since mScarlet is uniquely bound to a His tag.
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