pEZT-BM

The ability of oligomeric membrane proteins to assemble with subunits in varying functional proportions is a common feature of many systems. Recombinant expression of heterologous oligomeric proteins with well-defined stoichiometry facilitates detailed structural and functional analysis but remains a major challenge. Here, researchers present two methods to overcome this challenge: one for rapid virus titration and another for chemometric assays. When these methods are combined, they can effectively resolve heteromeric stoichiometry problems and optimize homogeneous protein expression. Using these two methods, they successfully expressed, purified, and grew diffraction-quality crystals of this challenging target.

In this study, the pFastBac Dual vector was used as the backbone for creating the pEZT-BM vector. In the vector, the researchers replaced the insect polyhedron promoter with a cassette containing the CMV promoter, multiple cloning sites, and transcriptional stabilizing elements. A synthetic, codon-optimized enhanced GFP gene (Bio Basic) was placed behind the insect p10 promoter in the opposite orientation to the CMV promoter. The p10 promoter drives expression of GFP in insect cells, allowing real-time monitoring of baculovirus production and subsequent virus titration. The CMV promoter and downstream stabilizing elements drive the expression of recombinant proteins in mammalian cells. The vector backbone contains the Tn7 transposable element for the bac-to-bac method of producing baculovirus. This vector was used to transiently transfect HEK cells and prepare bacmam virus. Creative Biogen can provide pEZT-BM vector.

Figure 1. pEZT-BM Vector and Its Use in Transduction. (Morales-Perez C L, et al. 2016)