Expression of soluble Type IV Minor Pilins and isolation of a Neisseria gonorrhoeae PilI-PilJ subcomplex

Abstract

Type IV pili and type II secretion systems assemble dynamic fibers used by bacteria and archaea for diverse functions. The pilus fiber is made up of major and minor pilin subunits containing a hydrophobic -helical spine and a globular head. Purifying minor pilins is complicated by the hydrophobic -helical spine, frequently present disulfide bonds, and low abundance within the fiber. These challenges have impeded structural and functional studies of pilin protomers. Here, we describe a method for expression and purification of soluble type IV pilin proteins from Escherichia coli. Signal peptidase I cleavage sites are engineered into the -helix of the pilin proteins. This allows their globular domains to be purified from the periplasmic fraction. We used this method to obtain the Neisseria gonorrhoeae minor pilins PilI and PilK in soluble form. In a third case, where the minor pilin PilJ could not be obtained on its own, coexpression with PilI and purification of a PilI-PilJ heterodimer was possible. We suggest that PilI and PilJ form an obligate heterodimer that is essential for their function. ImportanceType IV pili are essential to many bacteria responsible for disease. They can be found in both Gram-negative and Gram-positive bacteria, as well as archaea, making them likely present in the last common ancestor of all life on Earth. Despite their significance in a variety of species, there are large gaps in our understanding of the structure of these diverse biological machines. One roadblock to this research has been the difficulty of purifying the minor pilin proteins that serve different functions in the fiber. Here, we describe a novel method for the purification of these proteins and demonstrate the ability of this method to identify a protein-protein interaction between two minor pilins of Nesseria gonorrhoeae.