Therapeutic
To test whether our platform would be able to produce a therapeutic, we attempted to construct the human IL-10 encoding plasmid and express them in the gelated bacteria. We came to this specific protein through many talks with experts, about which you can read more on our Human Practices Page. However, expression of this protein was not straightforward, since IL-10 contains disulfide bridges that cannot be formed in the cytoplasm of bacteria. A targeting sequence to the periplasm is needed to correctly express this protein.
To this end, two gBlocks were designed, each with its own periplasmic targeting peptide, called the Ompf and PelB sequences, respectively[8]. This created the parts in the table below. These sequences had been used and tested in literature, also in combination with IL-10 expression. We decided to clone the genes in both the pBAD vector and the pET24a(+) vector as a backup. Initially, there were some struggles with cloning directly into the pBAD vector due to a small error in the gBLocks. Therefore, we first ligated it into pET24a(+), which was confirmed by a double digestion. These fragments were then purified and cloned into pBAD. This resulted in the following parts to be created:
| Construct | Name |
|---|---|
| pBad Ompf-IL10-His | BBa_K4905020 |
| pBad PelB-IL10-His | BBa_K4905021 |
Following cloning, all plasmids were sequenced to confirm the right insert was present.PBAD ompf-IL10 was transformed into E. coli BL21(DE3) and inoculated in TB medium. Protein expression was then induced and the products purified via periplasmic extraction, as described in Week 37 of the Notebook[9].
The periplasmic fraction of three different samples was loaded onto an SDS-PAGE gel (Figure 2). One sample was from a culture in which no arabinose was added, while the other two did contain arabinose and should therefore have expressed IL-10. However, we did not observe a band at the expected molecular mass of 18.6 kDa.
Although the expression of IL-10 was not sufficient to be observed by SDS-PAGE, we decided to perform an immunoassay on the samples anyway, because the assay has a very low detection limit. In this assay, we saw a clear difference between the measured sample and the control. However, the calibration curve did not look good enough to draw a definitive conclusion about the concentration of IL-10 expressed. This means that we were probably able to express a low concentration of IL-10, but it is questionable whether it was enough to act as a therapeutic in a real-life situation. The expression will need more optimization in the future.
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