Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers
OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium’s natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located in the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.
- Research Organization:
- Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
- Sponsoring Organization:
- USDOE
- Grant/Contract Number:
- 89233218CNA000001
- OSTI ID:
- 1492538
- Report Number(s):
- LA-UR-18-26823
- Journal Information:
- Langmuir, Vol. 33, Issue 38; ISSN 0743-7463
- Publisher:
- American Chemical SocietyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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