PeptiNanodisc: The Optimal Solution for Membrane Protein Cell Assays

Membrane proteins, especially multi-transmembrane proteins such as the GPCR family and ion channel proteins, play crucial roles in physiological processes including membrane transport, signal transduction, cellular homeostasis, and energy metabolism. Despite their importance, these proteins contain multiple hydrophobic regions, making it highly challenging to obtain stable, non-aggregated membrane proteins in their native conformation—one of the major obstacles in membrane protein-targeted drug development.

Detergents were the earliest solution for the expression and purification of full-length membrane proteins. As amphipathic molecules with both hydrophobic and hydrophilic regions, detergents can replace phospholipids and solubilize membrane proteins. However, detergents are inherently toxic and may disrupt the structure and activity of membrane proteins, thereby limiting their downstream applications. To overcome these issues, researchers developed a detergent-free alternative—Nanodiscs. Depending on the scaffold material used to stabilize the membrane proteins, Nanodiscs can be further categorized into Synthetic Nanodiscs and MSP (Membrane Scaffold Protein) Nanodiscs. MSP Nanodiscs are not suitable for downstream cell-based assays due to residual detergents. Similarly, Synthetic Nanodiscs are also not recommended for cellular experiments because their phospholipids are derived from natural cell membranes.

From an application-oriented perspective, DIMA Biotech has developed a simple and effective method—PeptiNanodisc—to stabilize membrane proteins without the use of detergents, after multiple rounds of optimization. As the name suggests, PeptiNanodisc utilizes specially designed peptides to wrap around the hydrophobic regions of membrane proteins (similar to a peptide-based disc), thereby protecting them from the aqueous environment.

Since it contains neither phospholipids nor detergents, membrane proteins expressed using the PeptiNanodisc platform are suitable for cell-based assays. Moreover, in theory, the peptide disc can act as a universal scaffold, applicable to a wide variety of membrane proteins regardless of their size.

The Advantages of PeptiNanodisc

Unlocks cell-based application scenarios (e.g., CAR positivity detection), addressing one of the major limitations of traditional Nanodiscs
Functionally active, validated by ELISA, with high bioactivity
Stable performance, suitable for lyophilization and shipping
Custom Service to meet customers’ demand: In addition to conventional proteins, fluorescently labeled versions such as FITC-labeled proteins are also being developed in parallel

The validated data of activity and stability The caseshow of FITC-PeptiNanodisc

PeptiNanodisc validated data

1. CAR Positivity Detection Application Data

CAR positivity rate is a key indicator for evaluating the efficacy of CAR-T products, with both the number and proportion being critically important. Whether in the preclinical or clinical stage, assessing CAR positivity is a fundamental aspect of CAR-T quality control. Currently, several methods are used to detect CAR positivity based on different CAR structural components, including Protein L, anti-Fab antibodies, anti-idiotype antibodies, target proteins, and anti-G4S linker antibodies. Among these, target proteins offer the highest specificity and can simultaneously assess the binding capability between the CAR and its target.

To detect CAR positivity using functional full-length membrane proteins, PeptiNanodisc is the optimal choice.

Comparison of CLDN18.2 CAR Positivity Detection Methods

CLDN18.2 PeptiNanodisc

Protein L-647

G4S-PE

Results showed that CLDN18.2 PeptiNanodisc (FLP420014) achieved comparable efficiency in detecting CLDN18.2 CAR positivity when compared to positive controls (Protein L-647 and G4S-PE). Since CLDN18.2 PeptiNanodisc binds to CLDN18.2 CAR through specific antigen–antibody interactions, it offers the highest specificity among all detection methods. Therefore, PeptiNanodisc represents the optimal solution for CAR positivity detection targeting corresponding membrane protein antigens.

Comparison of GPRC5D CAR Positivity Detection Methods

GPRC5D PeptiNanodisc

G4S-PE

Results showed that GPRC5D PeptiNanodisc (FLP400011) demonstrated comparable performance to the G4S-PE method in detecting GPRC5D CAR positivity.

2. Validation of Fluorescently Labeled PeptiNanodiscs

Labeled PeptiNanodisc, refers to PeptiNanodiscs containing full-length membrane proteins that are labeled. Unlike traditional labeled proteins, labeled PeptiNanodiscs are produced by first conjugating small molecules such as biotin or FITC directly onto the peptides, followed by assembly of the PeptiNanodisc. This process results in membrane proteins labeled with biotin or fluorescent tags.

CLDN18.2 CAR Positive Rate Determined by CLDN18.2 PeptiNanodisc-FITC

CLDN18.2 CAR Positive Rate Determined by G4S-PE

Results showed that FITC-labeled CLDN18.2 PeptiNanodisc demonstrated comparable detection of CLDN18.2 CAR positivity to the G4S-PE method, with the added advantage of requiring no fluorescent secondary antibody, making the procedure more convenient.

3 The Stability Data of PeptiNanodisc

Lyophilization and Storage Stability Testing of CLDN6-Strep PeptiNanodisc

Using functional ELISA, the binding activity of CLDN6-Strep PeptiNanodisc (FLP420008) to CLDN6 antibody was measured in both liquid and lyophilized powder forms. The results showed no significant difference in EC50.

Functional ELISA was used to assess the binding activity of CLDN6-Strep PeptiNanodisc to CLDN6 antibody under different storage conditions: -20℃, 37℃ for 24 hours, 37℃ for 48 hours, and 37℃ for 72 hours. The results showed no significant difference in EC50.

SDS-PAGE analysis showed that under different conditions (liquid state at -80℃, lyophilized powder at -20℃, and lyophilized powder stored at 37℃ for 24h, 48h, and 72h), the band position and intensity of CLDN6-Strep PeptiNanodisc exhibited no significant differences.

Lyophilization and Storage Stability Testing of GPRC5D PeptiNanodisc

Using functional ELISA, the binding activity of GPRC5D PeptiNanodisc to GPRC5D antibody was measured in both liquid and lyophilized powder forms. The results showed no significant difference in EC50.

Functional ELISA was used to assess the binding activity of GPRC5D PeptiNanodisc to GPRC5D antibody under different storage conditions: -20℃, 37℃ for 24 hours, 37℃ for 48 hours, and 37℃ for 72 hours. The results showed no significant difference in EC50 values.

SDS-PAGE analysis showed that under different conditions (liquid state at -80℃, lyophilized powder at -20℃, and lyophilized powder stored at 37℃ for 24h, 48h, and 72h), the band position and intensity of GPRC5D PeptiNanodisc exhibited no significant differences.