Recombinant protein expression is a prominent molecular technique for structural biology studies. For expressing recombinant proteins, a host organism is utilized as protein production factories. To obtain the desired recombinant proteins, we first design a recombinant DNA, which involves the information of protein to be produced. Then, we clone the desired DNA fragments into a system that assists the expression process. We call them “construct.”
A wide range of host organisms can be used for protein expression, ranging from bacteria and yeasts to animal (insect/mammalian) and even plant cells. However, bacterial expression systems have many advantages over other host systems as they allow high-level expression at low cost. Therefore, we prefer testing bacterial cells (Escherichia coli) first before moving to more complex systems.
Here, in this post, we share all the constructs tested so far by me, Cheng Dong, and Lei Ming to produce a high amount of soluble SETDB1 protein. We have tested more than 80 constructs that correspond to different regions of the catalytic domain of SETDB1 protein. Out of these 80 trials, only 3 constructs have shown a positive result for expression. We listed the details of the constructs tested in this Zendo post.
In our next experiments, we plan to focus on improving the purity of proteins obtained by these 3 constructs. In addition to the optimization of purification process, we will also characterize the stability of resulting proteins before toward the efforts aimed at structural elucidation.