Membrane proteins account for approximately 20-30% of proteins encoded by the human genome and play central roles in cell signaling, substance transport, and cell recognition. Aberrant expression and dysfunction of membrane proteins are closely associated with a variety of diseases, including cancer, neurological disorders, and metabolic diseases, making them key focuses in drug discovery.
Multi-pass membrane proteins are widely found in receptor families (such as GPCRs), ion channels, transporters, and membrane-associated enzymes. These proteins play pivotal roles in processes such as signal transduction, material transport, and cell recognition, and represent important drug targets. In particular, multi-pass membrane proteins such as G protein-coupled receptors (GPCRs), ion channels, and transporters account for more than 60% of FDA-approved drug targets.
However, due to their strong hydrophobicity, complex structures, and strict dependence on native environments, the preparation of full-length and functionally intact multi-pass membrane proteins has long been a major technical challenge in biopharmaceutical research and development.
1. Challenges in Preparing Full-Length Multi-Pass Membrane Proteins
Full-length multi-pass membrane proteins (e.g., GPCRs, ion channels, and ABC transporters) possess complex tertiary and quaternary structures with multiple transmembrane helices. Their native conformations rely heavily on the membrane environment. The main challenges include:
Difficult expression: Due to structural complexity, these proteins often misfold or form inclusion bodies in heterologous expression systems, leading to low yields and poor functional integrity.
Challenging purification: A large amount of non-membrane components must be removed while maintaining protein stability. Conventional detergents frequently disrupt the structure and activity of membrane proteins.
Limited structural analysis: Multi-pass membrane proteins are difficult to crystallize and require a membrane-like environment. Although cryo-EM has made significant progress, sample preparation remains highly challenging.
Complex functional reconstitution: Functional assays require a membrane-mimicking environment, as conventional aqueous systems fail to replicate the native membrane state.
To overcome bottlenecks in the expression, purification, and stabilization of full-length membrane proteins—particularly those requiring a membrane-like environment due to their multi-pass structures—researchers have developed the Nanodisc preparation platform. This system constructs a stable artificial or semi-artificial lipid bilayer microenvironment, encapsulating membrane proteins in a near-native state. Nanodiscs not only greatly enhance protein stability and activity but also provide an ideal scaffold for structural analysis, functional studies, and drug screening.
Currently, Nanodisc technology has evolved into several formats, including membrane scaffold protein (MSP) Nanodiscs, Synthetic Nanodiscs, and PeptiNanodiscs, each offering unique advantages and applications.
2. What Types of Nanodiscs Are There?
Based on the stabilizing agents used, Nanodiscs can be categorized into different types: MSP Nanodiscs, Synthetic Nanodiscs, and PeptiNanodiscs.
2.1 MSP Nanodisc
MSP nanodiscs use membrane scaffold proteins (MSPs) as stabilizers to encapsulate artificial phospholipid components and transmembrane proteins, thereby forming a nanoscale disc. The size of the nanodisc formed is determined by the MSPs used, typically ranging from 7 to 13 nm in diameter. Commonly used MSPs in nanodisc assembly include MSP1D1, MSP1D1-DH5, and MSP1E3D1. These MSPs have been extensively studied and proven effective in generating stable and functional nanodisc proteins.
Fig 1. Assembly of MSP-Nanodisc
2.2 Synthetic Nanodisc
Synthetic Nanodiscs are produced using synthetic polymers, typically styrene–maleic acid copolymer (SMA) and diisobutylene–maleic acid (DIBMA). The assembly of synthetic nanodiscs starts directly from intact cells. Synthetic polymers solubilize the cell membrane while native phospholipids form nanodisc structures around the membrane proteins. In this process, the polymers act simultaneously as solubilizers and stabilizers. Therefore, no additional detergents are required.
Fig 2. Assembly of Synthetic Nanodisc
2.3 PeptiNanodisc
PeptiNanodisc is a technology that uses specialized peptides to wrap around the hydrophobic regions of membrane proteins (similar to peptide discs), thereby protecting them from the aqueous environment. Since neither phospholipids nor detergents are involved, membrane proteins expressed using the PeptiNanodisc platform can be directly applied in cell-based assays. Moreover, in theory, peptide discs can function as a “universal scaffold,” adaptable to a wide variety of membrane proteins without being restricted by protein size. This technology enables the stable reconstitution of multiple classes of membrane proteins and is particularly suitable for presenting transmembrane targets in their native conformations.
Fig 3. Assembly of PeptiNanodisc
3. Advantages and Applications of Nanodiscs
3.1 Membrane Protein Structure and Function Studies
- Advantages:
High stability of native conformation: Nanodiscs provide a lipid bilayer environment similar to cell membranes, maintaining the natural conformation and functional activity of membrane proteins.
High homogeneity and controllability: Lipid composition can be customized, allowing the simulation of diverse membrane microenvironments.
Elimination of micelle interference: Compared with detergents, Nanodiscs cause less structural disturbance to proteins, making them suitable for high-resolution structural analysis.
- Applications:
Cryo-EM: High-resolution structural determination of GPCRs, ion channels, transporters, and more.
NMR, SPR, ITC, and other biophysical studies: Precise measurement of binding kinetics between membrane proteins and ligands or antibodies.
3.2 Drug Target Discovery and Validation
- Advantages:
Native presentation of targets: Membrane proteins in Nanodiscs closely mimic the conformation found in cell membranes, facilitating the discovery of true binding molecules.
High sensitivity in ligand screening: Suitable for high-throughput screening of small molecules, antibodies, and peptide drugs.
Accurate antibody epitope mapping: Minimizes false negatives caused by protein denaturation.
- Applications:
Lead compound screening: Ligand binding assays on GPCR Nanodiscs to identify high-affinity lead molecules.
Antibody screening and optimization: Nanodiscs combined with flow cytometry or BLI/SPR to select antibodies with high specificity and functional activity.
3.3 Vaccine and Immunogen Design
- Advantages:
Preservation of key conformational epitopes: Ideal for presenting conformationally sensitive antigens such as viral envelope proteins and bacterial outer membrane proteins.
Tunable lipid environment: Enhances immunogen stability and immune response.
- Applications:
Vaccine development: Display of HIV Env, SARS-CoV-2 S protein, and others on Nanodiscs to induce neutralizing antibody responses.
3.4 Detection and Diagnostics
- Advantages:
Enhanced sensitivity of immunoassays: Membrane proteins maintain their native conformation on Nanodiscs, improving assay accuracy.
Quantitative detection of difficult-to-solubilize proteins: Overcomes challenges of membrane protein instability and precipitation.
- Applications:
Detection of membrane protein–specific antibodies using ELISA, flow cytometry, and Luminex platforms.
Companion diagnostics: Measuring patient serum antibody responses against target proteins.
3.5 Biologics Quality and Functional Assessment
- Advantages:
Antibody functional validation: Direct evaluation of antibody binding and blocking activity on native conformation targets.
Drug stability testing: Assessment of the thermal stability and tolerance of antibody/ligand–membrane protein interactions.
- Applications:
ADC internalization assays (e.g., DT3C simulation platform).
CAR-T cell positivity rate assessment.
Thermal stability analysis of antibody interactions with GPCRs or ion channels.
4. Advantages of DIMA Nanodisc Products
The DIMA Nanodisc full-length membrane protein production platform possesses a globally leading membrane protein library, with over 500 types of full-length multi-transmembrane proteins available in stock, including GPCRs, ion channels, and other important drug targets. Technically, the platform has successfully produced full-length membrane proteins with up to 24 transmembrane domains, setting a new industry record. Product validation includes SDS-PAGE, ELISA, SPR, HPLC, etc., ensuring high purity, high solubility, and high stability, while maintaining native conformation and supporting room-temperature transport.
Additionally, the platform provides flexible, customized protein expression and production services to meet diverse research needs.
Validation Data Display
- SDS-PAGE
- ELISA
- SEC-HPLC
- SPR
- Detection of CAR-T cell positivity rate
GPRC5D PeptiNanodisc (Cat.No.FLP400011) and G4S-PE protein were used to detect the positivity rate of GPRC5D CAR-T cells.
FITC-labeled CLDN18.2 PeptiNanodisc (Cat.No.FLP420014) and G4S-PE protein were used to detect the positivity rate of CLDN18.2 CAR-T cells.
