Nanodiscs: A Versatile Platform for Membrane Protein Research

Membrane proteins are essential to numerous cellular processes and represent more than half of all current drug targets—yet they remain some of the most difficult proteins to study in vitro due to their dependence on the lipid bilayer. Traditional approaches often rely on detergents to extract and solubilize membrane proteins, which can compromise their structure, function, or stability. Nanodiscs offer a powerful alternative: they provide a soluble, nanoscale lipid bilayer environment that preserves membrane protein conformation while enabling structural and functional analyses. In this article, we explore the fundamentals of nanodisc technology, including how they are produced, the different scaffold systems available, and how KACTUS’ proprietary copolymer nanodiscs simplify membrane protein research and screening.

What is a Nanodisc?

Nanodiscs are synthetic, nanoscale disc-shaped lipid bilayers that provide a stable, soluble mimetic of cell membranes for studying membrane proteins. Structurally, a nanodisc consists of a phospholipid bilayer (typically ~10 nm in diameter) whose hydrophobic perimeter is stabilized by amphipathic molecules – either membrane scaffold proteins (MSPs) or synthetic copolymers. In classical MSP-based nanodiscs, two copies of an engineered protein wrap around the lipid bilayer like a belt. Newer “polymer nanodiscs” use amphipathic copolymers (e.g. styrene–maleic acid or SMA) instead of protein belts to solubilize a lipid patch in the absence of detergents. In both cases, the result is a water-soluble, discoidal lipid bilayer particle that can incorporate membrane proteins in a near-native environment [1].

Figure 1. Structure of a transmembrane protein inside a copolymer nanodisc.  

Nanodisc Types & Production

MSP-Based Nanodiscs

The original nanodisc platform uses membrane scaffold proteins (MSPs) to stabilize the lipid disc. MSPs are typically truncated apolipoprotein A-I variants (~200 amino acids) that self-assemble around a lipid patch. To assemble an MSP nanodisc, a purified membrane protein is first solubilized in a suitable detergent along with phospholipids. MSP protein is then added, and the detergent is removed (for example, by dialysis or hydrophobic beads), triggering the MSPs and lipids to coalesce into disc-shaped nanostructures with the target protein embedded. Each nanodisc is formed by two MSP molecules wrapping around the lipid bilayer. The diameter of the disc is determined by the length of the MSP belt and the lipid:MSP ratio used. This self-assembly process often produces a mixture of “empty” nanodiscs (without protein) and protein-loaded nanodiscs, which can be separated by size-exclusion or affinity chromatography [2]. MSP nanodiscs have been widely adopted because they produce monodisperse, stable lipid bilayer particles for a broad range of membrane proteins.

Figure 2. Schematic of two methods for assembling membrane proteins (MPs) into nanodiscs. Route 1 (standard) involves detergent solubilization of purified MP, MSP, and lipids, followed by detergent removal. Route 2 (alternative) directly solubilizes membrane/tissue with lipids and MSP, rapidly removing detergent to form nanodiscs containing various MPs. Target MP is then purified while stabilized within the nanodisc, which can also generate a membrane protein library [3].

Amphipathic Peptide-Based Nanodiscs

A newer approach involves short synthetic amphipathic peptides that mimic the structural role of scaffold proteins. These peptides, such as apolipoprotein-A-I sequences, self-assemble with phospholipids to form nanodiscs, typically following a similar process as MSP-based systems: the membrane protein is co-solubilized with lipids and detergent, peptides are added, and detergent is removed to initiate nanodisc formation. Peptide nanodiscs are chemically defined, tunable, and smaller in size (~6–8 nm), making them attractive for certain NMR and biophysical applications. Compared to MSPs, peptides are easier to synthesize and modify, allowing greater customization. However, peptide nanodiscs often exhibit lower stability and may not support the incorporation of large or complex membrane proteins as effectively. This platform remains under active development, with growing interest for high-throughput screening and targeted studies of small membrane proteins [4]

Polymer-Based Nanodiscs

A novel approach uses synthetic amphipathic copolymers – often styrene–maleic acid (SMA) or more recently DIBMA and related polymers – to extract membrane proteins in the form of native nanodiscs (sometimes called SMA lipid particles or SMALPs). These polymer nanodiscs are formed by adding the copolymer directly to cell membranes, where it inserts into the lipid bilayer and excises a nanoscale disk of lipid membrane containing the target protein. The polymer wraps around the perimeter of the excised membrane patch (in place of an MSP), yielding a lipid–polymer nanodisc that contains the protein and its surrounding native lipids. This process does not require any detergent, since the polymer acts as both the solubilizing agent and the stabilizer. The polymer nanodiscs are purified using affinity chromatography. 

Figure 3. SMA copolymers extract membrane proteins along with native lipids to form nanodiscs. The resulting mixture includes both protein-loaded and empty nanodiscs, which can be separated by affinity purification to isolate nanodiscs containing the target protein [5]

A major advantage is that the target’s native lipid environment is retained in the nanodisc, which can be critical for preserving function. Polymer-based nanodiscs also avoid the trial-and-error of finding a compatible detergent and lipid mixture, simplifying the development cycle for challenging membrane proteins. However, polymer approaches have their own considerations: for example, SMA copolymers are sensitive to divalent cations and low pH, and the styrene components absorb UV light (which can interfere with protein assays) [6]. 

KACTUS has developed a proprietary copolymer to streamline the production of membrane protein nanodiscs. This proprietary amphipathic copolymer is added directly to the cell membranes to solubilize the proteins into nanodiscs and ensures production quality, performance, and reliability. 

Figure 3. Expression of KACTUS copolymer nanodiscs. Membrane proteins are expressed in HEK293 mammalian cells. The addition of copolymers results in self-assembly of discoidal lipid bilayers which are then purified. 

Applications of KACTUS Copolymer Nanodiscs

Nanodiscs have opened up a wide range of applications in membrane protein research, drug discovery, and structural biology. By providing a stable lipid bilayer, nanodiscs enable solubilized membrane proteins to be studied in their native structure and function. Below are some of the major applications of nanodisc technology:

• Yeast display screening

• Antibody Panning

• CAR expression testing via FACS

• PK/PD Studies

• Analytical Assays, ELISA, SPR, BLI

Advantages of KACTUS Copolymer Nanodiscs

• Native Lipid Environment - KACTUS nanodiscs incorporate the native cellular phospholipids that were present around the protein in the cell membrane, not artificial lipids

• Toxic transmembrane proteins - Nanodiscs can incorporate toxic transmembrane proteins, making them a flexible tool for studying challenging proteins

• High Purity - KACTUS nanodiscs are affinity purified, ensuring homogenous complexes and no “empty” nanodiscs

• Detergent-Free Extraction - Our nanodiscs are expressed completely detergent-free to preserve native activity

Nanodisc Membrane Protein Catalog

KACTUS offers an off-the-shelf catalog of membrane proteins presented on copolymer nanodiscs, as well as custom nanodisc protein expression services. Our catalog products cover a variety of challenging targets – GPCRs, ion channels, transporters, and other multipass proteins. By leveraging its copolymer nanodisc platform, KACTUS can supply full-length, properly folded membrane proteins for use in research or screening. These nanodiscs are available in biotinylated and non-biotinylated formats and undergo quality control testing for purity and bioactivity using SPR and ELISA assays. 

Product Performance Validation

Human CCR8 Protein-Nanodisc (CR8-HM1N29)

Human CCR8 nanodisc, His Tag captured on CM5 Chip via Anti-His Antibody can bind Anti-CCR8 Antibody, hFc Tag with an affinity constant of 53.40 pM as determined in SPR assay (Biacore T200).

Human CCR8 Protein-Nanodisc (CR8-HM1N29)

Immobilized Biotinylated Human CXCR5 Nanodisc, His Tag at 5ug/ml (100ul/well) on the streptavidin precoated plate (5ug/ml). Dose response curve for Anti-CXCR5 Antibody, hFc Tag with the EC50 of 18.1ng/ml determined by ELISA (QC Test).

Biotinylated Human Claudin 4 Protein-Nanodisc (CLD-HM14NB)

Biotinylated Human Claudin 4 Nanodisc, His Tag captured on CM5 Chip via Streptavidin can bind Anti-Claudin 4 Antibody, hFc Tag with an affinity constant of 6.145 nM as determined in SPR assay (Biacore T200).

Biotinylated Human GLP-1R Protein-Nanodisc (GLP-HM4N185BF)

Immobilized Biotinylated Human GLP-1R Nanodisc, His Tag at 5ug/ml (100ul/well) on the streptavidin precoated plate (5ug/ml). Dose response curve for Anti-GLP-1R Antibody, hFc Tag with the EC50 of 0.28ug/ml determined by ELISA (QC Test).

Product List

References

1. Sligar, S. G., & Denisov, I. G. (2021). Nanodiscs: A toolkit for membrane protein science. Protein science : a publication of the Protein Society, 30(2), 297–315. https://doi.org/10.1002/pro.3994

2. Hagn, F., Nasr, M. L., & Wagner, G. (2018). Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nature protocols, 13(1), 79–98. https://doi.org/10.1038/nprot.2017.094 

3. Denisov, I. G., & Sligar, S. G. (2016). Nanodiscs for structural and functional studies of membrane proteins. Nature structural & molecular biology, 23(6), 481–486. https://doi.org/10.1038/nsmb.3195

4. Salnikov, E. S., Anantharamaiah, G. M., & Bechinger, B. (2018). Supramolecular Organization of Apolipoprotein-A-I-Derived Peptides within Disc-like Arrangements. Biophysical journal, 115(3), 467–477. https://doi.org/10.1016/j.bpj.2018.06.026

5. Dörr, J. M., Scheidelaar, S., Koorengevel, M. C., Dominguez, J. J., Schäfer, M., van Walree, C. A., & Killian, J. A. (2016). The styrene-maleic acid copolymer: a versatile tool in membrane research. European biophysics journal : EBJ, 45(1), 3–21. https://doi.org/10.1007/s00249-015-1093-y

6. Hawkins, O. P., Jahromi, C. P. T., Gulamhussein, A. A., Nestorow, S., Bahra, T., Shelton, C., Owusu-Mensah, Q. K., Mohiddin, N., O'Rourke, H., Ajmal, M., Byrnes, K., Khan, M., Nahar, N. N., Lim, A., Harris, C., Healy, H., Hasan, S. W., Ahmed, A., Evans, L., Vaitsopoulou, A., … Rothnie, A. J. (2021). Membrane protein extraction and purification using partially-esterified SMA polymers. Biochimica et biophysica acta. Biomembranes, 1863(12), 183758. https://doi.org/10.1016/j.bbamem.2021.183758