PURIFICATION AND CHARACTERIZATION OF P97 AND LIPID NANODISC
Date
2025-05
Authors
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Publisher
University of Delaware
Abstract
p97’s main function in the cell is to act as a segregase, isolating misbehaving
proteins from their environment. Importantly, p97 plays this role in Endoplasmic
Reticulum-associated degradation (ERAD), a cellular pathway that removes deviant
proteins, whether they be misfolded, misbehaving, or misplaced, from the ER. For p97
to perform this role, it must be localized to the ER membrane. This job of localizing
p97 can be done by selenoprotein S, a protein embedded into the ER membrane.
Yet there are still many unanswered questions about the details of this interaction.
If we want to study this interaction, we need to purify and characterize p97 and a
membrane mimic for selenoprotein S to embed in. We expressed p97 in Rosetta2 (DE3)
cells and purified p97 with immobilized metal affinity chromotgraphy. Moreover, we
characterized p97 assembly with size exclusion chromatography.
We choose to use lipid nanodiscs as our membrane mimic. Lipid nanodiscs
are composed of a lipid bilayer surrounded by a long helical protein called membrane
scaffolding protein (MSP). We expressed MSP in BL21 (DE3) cells and purified MSP
with a series of nickel charged columns. Lastly, we assembled the nanodisc with 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids and imaged the nan odisc with Negative Staining Transmission Electron microscop