High-Level Prokaryotic Expression and Purification of Death Domain Superfamily with MBP Tag

Death domain superfamily are compact structural motifs that play critical roles in many biological processes including inflammation and apoptosis. Due to mediating protein-protein interactions, these death domains have a high tendency to form oligomers and are notoriously difficult to deal with when overexpressed in vitro. In this study, we found that maltose binding protein (MBP) is very effective in improving the behavior of many death domain superfamily members.

In order to achieve high-level expression of death domain superfamily in E. coli, we designed two MBPtagged expression vectors based on a pET30a backbone: one with a short flexible noncleavable linker, the other with a TEV cleavage site. Soluble protein was purified from cell lysate by HisTrapTM IMAC column and Superdex-200 gel filtration column.

We tested seven targets of death domain superfamily, including two PYDs, three CARDs, and two DDs; for all of these, it is challenging to obtain recombinant protein without a tag. We expressed and purified these death domain proteins successfully as soluble and highly purified protein with both vectors. Among them, two proteins were crystallized successfully.

Our study demonstrated that both recombinant MBP expression vectors significantly enhanced production. In addition, MBP tagged recombinant proteins can sometimes produce crystals. This strategy may be applied to other challenging proteins.