(P14-LB) Cell-free screening and production of chlamydial membrane bound proteins

Background: Membrane proteins relevant as vaccine antigens for sexually transmitted infections (STIs) often pose several production-related difficulties, including insolubility and tendency towards aggregation. This postures a significant obstacle to development of vaccines for common STIs such as Chlamydia, Syphilis and Gonorrhea that continue to impact millions with chronic health issues.

Methods: Small scale screening (nL-uL) identified solubilized membrane proteins that have inserted into the nanodisc. We then applied a medium scale screen (1-3 mL) for characterizing protein yield and conducted biochemical as well as structural analyses. Functional testing relied on protein-protein/lipid/antibody interactions and electrophysiology. Structural analyses used size exclusion chromatography, atomic force microscopy and electron microscopy.

Results: Combined with nanolipoproteins particles, 10-25 nm membrane mimetic nanodiscs, we can quickly produce and purify chlamydial-related membrane bound antigens of interest. Using this approach, we can screen, solubilize, and scale-up multiple proteins, including variants of the major outer membrane protein (MOMP), polymorphic membrane proteins (Pmps), type three secretion systems, inclusion membrane proteins (Inc) and others.

Conclusions: Cell free protein synthesis technology provides a systematic method of high-throughput membrane protein and protein complex production. Current efforts are focused on looking at inclusion membrane protein A (Inc-A) and Ygg for testing alone and in combination with MOMP in future animal challenge studies. This approach will benefit the community through development of STI-related vaccines using functionally folded membrane bound proteins.