In my previous post, I shared the expression and purification results of several constructs based on bacterial expression systems. Such experiments often fail as there are inherent complications with protein expression associated with bacterial expression systems. This was likely the case for most of our last batch samples. To tackle this problem, we decided to switch a more complex protein production machinery, insect cells.
Although there are downsides of insect cells, higher costs and longer duration of culture growth (2-3 weeks), they have many advantages over bacterial cells. They can perform post-translational modifications (some sort of chemical marks on the proteins) and, folding of mammalian originated proteins. These increase the likelihood of expression and improve the solubility of end-products, proteins. Therefore, important to note that proteins produced by insect cells are also often more functional than those produced in bacterial systems.
In our initial trials, we had made four different constructs exploiting a commonly used insect cell platform called Sf9. (“Sf” stands for the initials of “Spodoptera frugiperda.”) Unfortunately, these initial trials also did not provide a positive result for the expression of SETDB1 catalytic domain. The details of samples and results are available in this Zenodo post. We will continue to test Sf9 cells to identify well expressing and soluble SETDB1 constructs and keep sharing our progress here.