Purification and characterization of Shigella Invasion Plasmid Antigen C
Class
Article
Department
Chemistry and Biochemistry
Faculty Mentor
Nicholas Dickenson
Presentation Type
Poster Presentation
Abstract
Invasion Plasmid Antigen C (IpaC) is a critical member of the type three secretion system (T3SS) expressed by Shigella spp. It plays a dual role in serving as both a structural component and potent effector of the T3SS. Structurally, IpaC resides at the tip of the mature T3SS needle, completing the translocon pore through which effectors are secreted from the bacterial cytoplasm into the host cell. As an effector, IpaC is injected into the host cell, polymerizing actin near the host membrane and inducing macropinocytotic uptake of the pathogen. Understanding the precise roles and interactions of IpaC with both Shigella and host cell components is of great interest, however, the hydrophobic nature of IpaC has proven a challenge in expressing and purifying the recombinant protein. Here, this was overcome by co-expressing and co-purifying IpaC with its cognate chaperone IpgC. This approach allowed purification of large quantities of the complex with a high degree of purity and, following a series of stability assays in varying buffer conditions, crystallization trays have been set up in hopes of obtaining the first structure of a chaperone bound T3SS effector. Furthermore, several detergent candidates were explored as a means of separating IpaC from the purified complex, providing additional capabilities to study the protein absent of the chaperone. Together, these results have produced the ability to purify, isolate, and study IpaC in both its chaperone bound and "free" states, providing a platform for ongoing work with this important T3SS protein.
Start Date
4-9-2015 3:00 PM
Purification and characterization of Shigella Invasion Plasmid Antigen C
Invasion Plasmid Antigen C (IpaC) is a critical member of the type three secretion system (T3SS) expressed by Shigella spp. It plays a dual role in serving as both a structural component and potent effector of the T3SS. Structurally, IpaC resides at the tip of the mature T3SS needle, completing the translocon pore through which effectors are secreted from the bacterial cytoplasm into the host cell. As an effector, IpaC is injected into the host cell, polymerizing actin near the host membrane and inducing macropinocytotic uptake of the pathogen. Understanding the precise roles and interactions of IpaC with both Shigella and host cell components is of great interest, however, the hydrophobic nature of IpaC has proven a challenge in expressing and purifying the recombinant protein. Here, this was overcome by co-expressing and co-purifying IpaC with its cognate chaperone IpgC. This approach allowed purification of large quantities of the complex with a high degree of purity and, following a series of stability assays in varying buffer conditions, crystallization trays have been set up in hopes of obtaining the first structure of a chaperone bound T3SS effector. Furthermore, several detergent candidates were explored as a means of separating IpaC from the purified complex, providing additional capabilities to study the protein absent of the chaperone. Together, these results have produced the ability to purify, isolate, and study IpaC in both its chaperone bound and "free" states, providing a platform for ongoing work with this important T3SS protein.