First developed by Stephen G. Sligar, the nanodisc is a phospholipid bilayer surrounded by a belt of an engineered apolipoprotein A1 derivative. By manipulating the lipids used as well as the protein belt, one can change the diameter of the disc and reconstitute a target membrane protein into this synthetic membrane system. Considered a more native environment than the amphipol or detergents micelles commonly used for membrane protein studies, nanodisc reconstitutions are of particular interest in the structural studies of membrane proteins.
In principle a nanodisc reconstitution is a simple procedure wherein the target protein is extracted and purified in detergent, followed by incubation with lipids and membrane scaffold protein (MSP). The detergent is then removed, permitting the reconstitution of the target protein into the nanodisc. In practice there are many routes to failure. The ratio of protein:lipid:nanodisc as well as the types of lipids to employ are often unknown and must be empirically determined.
What follows is a generalized protocol for reconstituting a channel in MSP2N2. Several, in-depth protocols exist for reconstitution, and an essential primer on the subject is found here.
Bacterial expression of these proteins can be found here.
The protocol can take a full, eight hours and should be performed the day of extraction. It is extremely helpful to prepare as many materials as possible a day in advance.
Materials
| Catalog Number | Notes | |
|---|---|---|
| MSP2N2 | 29520 | Stored at -80C in flash frozen aliquots of 100uL |
| Soy Polar Lipids | 541602 | In chloroform |
| N-Pentane | 8209571000 | Stored under hood, handle with caution |
| Lipid Extruder Set | 610000 | Can also use sonicator or combine both sonicator and extruder |
| PE Drain Disc (10nm) | 230300 | Cheaper to buy through Cytiva as opposed to Avanti |
| PC Membrane (0.2uM) | 610006 | Membrane size is rarely recorded in literature, but I assume there is variability. I have also used the 0.1uM membrane for lipid extrusion. |
| Bio-Beads SM-2 Resin | 1528920 | Aliquots stored at 4C are useable two months after initial preparation. Should be prepared in advance if possible. |
| Argon Gas | Next to fume hood | |
| 70% Denatured Alcohol | ||
| 150mM NaCl / 50mM Tris pH 8.0 (TBS) | ||
| MilliQ Water |
Extract target protein per usual protocols. During extraction step, begin the process of lipid extrusion.