Purification of the Outer Membrane Usher Protein and Periplasmic Chaperone-Subunit Complexes from the P and Type 1 Pilus Systems

Understanding molecular mechanisms of protein secretion by bacteria requires the purification of secretion machinery components and the isolation of complexes between the secretion machinery and substrate proteins. Here, we describe methods for the purification of proteins from the chaperone/usher pathway, which is a conserved secretion pathway dedicated to the assembly of polymeric surface fibers termed pili or fimbriae in gram-negative bacteria. Specifically, we describe the isolation of the PapC and FimD usher proteins from the bacterial outer membrane, and the purification of PapD-PapG and FimC-FimH chaperone-�subunit complexes from the periplasm. These Pap and Fim proteins belong to the P and type 1 pilus systems of uropathogenic Escherichia coli , respectively.