In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

ABSTRACT

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of ß-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented ß-barrel membrane proteins via intact ß-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled ß-barrel membrane proteins by demonstrating four representative ß-barrel membrane proteins E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.