Manish Kumar and Andrew Zydney

Membrane Filtration of Membrane Protein Detergent Mixtures

Membrane proteins (MPs) carry out distinct physiological functions in living organisms where they play important roles in the transport of substances into and out of cells, catalysis of metabolic reactions, and response to external stimuli. MPs make up one third of the lipid bilayer by weight and are targets for a large fraction, about 70%, of commercially available drugs. Thus there is significant interest in deciphering the mechanism of action and structural features of membrane proteins to design drugs with greater specificity and efficacy. Despite the growing interest in membrane proteins, they only account for about 2% of the protein structures reported in Protein Data Base (PDB). The primary challenge in working with membrane proteins arises from difficulties in extracting MPs in structurally and functionally stable forms.. The hydrophobic exterior of MP necessitates their stabilization in amphipathic detergent micelles. Another challenge with MP characterization arises from relatively low yields. Ultrafiltration using commercially available centrifugal concentrators is employed for the concentration of MPs but it is accompanied by concomitant concentration of detergentmolecules. This complicates subsequent characterization of MPs and frequently leads to precipitation and denaturation. Additionally many processes require removal of detergents by cumbersome processes such as dialysis or hydrophobic adsorption.

This REU project combines both fundamental and applied aspects of biomolecular separations relevant to the pharmaceutical industry. The REU student will learn about membrane protein purification and then carefully study the effect of pressure on comparative passage of detergent and membrane protein across various commercial ultrafiltration membranes. These results will be compared with centrifugal separations and guidance and models developed to guide the development of effective membrane protein concentration strategies.

rendering of a complex molecule

Explicit detergent molecules associated to a low-resolution model of FhaC, the Bordetella pertussis outer-membrane representative of the Omp85 superfamily of bacterial protein transporters. Image courtesy: Biophysical Journal Cover from July 2014 issue.

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Contact Information

Manish Kumar, Ph.D.
Assistant Professor of Chemical Engineering
REU Program Coordinator

Esther Gomez, Ph.D.
Assistant Professor of Chemical Engineering
REU Program Coordinator

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