2024 AIChE Annual Meeting
Purification and Self-Assembly of Outer Membrane Protein F
Membrane proteins are very promising targets for designing selective ion and water purification processes because of their high selectivity. In this work, we describe the purification and self assembly of wildtype Outer Membrane Protein F (WT OmpF) and one of its variants designated as PARA OmpF. WT OmpF has a pore size of 1.08 nm2 while PARA OmpF has been engineered to have a pore size of 0.3 nm2 by the insertion of bulky residues on the pore wall. Identical purification methods consisting of cell lysis, multiple ultracentrifugation and solubilization steps, and two rounds of chromatography were used to purify both variants of OmpF. Post-purification analysis using an SDS-Page Gel and the Bradford Assay revealed concentration and relative purity differences between WT and PARA OmpF despite the use of the same purification methods. Both purified and detergent solubilized OmpF variants were mixed with poly(butadiene-b-ethylene oxide) (PB-PEO) in an attempt to induce self-assembly into planar 2D nanosheets using detergent dialysis. The resulting materials were analyzed with transmission electron microscopy which revealed a high and ordered packing of WT OmpF, indicating its potential for rapid and selective transport while the PARA OmpF had formed vesicle structures. We hypothesize that lower polymer to protein ratios may induce successful 2D nanosheet formation in the future. The successfully created WT OmpF 2D nanosheets can then be deposited onto a polymer support with any gaps being filled in by an amphiphilic lipid to create a biomimetic membrane similar in structure to human skin. This skinlike biomimetic membrane displays incredible vapor transport properties compared to other breathable fabrics despite having a pore size that is multiple magnitudes of order lower. Ultimately, the purification and self assembly of WT OmpF and its variants is an important first step in creating novel materials with unique transport properties.