KACTUS: Transmembrane Proteins

Background

KACTUS has developed two transmembrane protein display platforms: virus-like particles (VLPs) and nanodiscs. These platforms are designed for functional display of multipass transmembrane proteins in their native conformation. KACTUS produce these full-length membrane proteins using detergent-free extraction methods, which preserve the structural integrity and biological activity of the proteins. Their products are suitable for a range of applications including immunisation, ELISA, yeast display, flow cytometry, and SPR. For these applications, choose from unlabelled, biotinylated, or fluorescent proteins. Additionally, their transmembrane proteins undergo bioactivity and purity testing using SPR, ELISA, flow cytometry, and HPLC. This ensures functional performance and batch consistency for reproducible results. Researchers can apply these products to antibody discovery, antibody screening, analytical method development, and immunisation.

Membrane Protein Technology Platforms

Virus-Like Particle (VLP)

Copolymer Nanodisc

Mammalian-expressed transmembrane proteins displayed on VLPs for native conformation and boosted immunogenicity

Transmembrane proteins expressed on membrane-like structures composed of phospholipids and copolymers

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Virus-like Particle (VLP) Proteins

Virus-like particles (VLPs) are small nanoparticles composed of virus shell or capsid proteins. These proteins self-assemble into virus-like structures, mimicking the organisation and morphology of the actual virus. However, VLPs are absent of viral infectious genomes, making them non-infectious and safe for use in various applications, including antibody immunisation, screening, vaccine development and gene therapy.

Diagram showing virus-like particle (VLP) expression with full-length transmembrane protein for antibody discovery — *Mechanism of expression for VLP-displayed antigens.*

Advantages of VLP Proteins

VLPs display the target protein in a dense manner on their surface. This boosts the immune response to the target protein, making VLPs useful for immunisation, especially with antigens that are usually present in low amounts or don’t trigger a strong immune reaction by themselves. VLPs also allow for the soluble expression of multipass transmembrane proteins without the use of detergents.

Applications

Blood sample determination: ELISA
In vivo pharmacokinetic analysis
Antibody immunisation, screening, and functional characterisation
CMC method development
Affinity determination via ELISA, SPR

Features

Site-Specific Biotinylation
VLP capsid proteins optimised for each protein
Proven biological activity via ELISA and SPR
Mammalian cell expression system for natural folding and glycosylation
Boosted immunogenicity for antibody drug discovery

Product Validation Data

Claudin 18.2 VLP

KACTUS was the first company to express full-length, wild-type Claudin 18.2. This complex 4-transmembrane domain protein has therapeutic potential for gastric and esophageal adenocarcinomas. Despite interest in developing drugs aimed at Claudin 18.2, technical challenges in expressing high-quality Claudin 18.2 protein had limited the potential for antibody drugs around this target. Quality recombinant target proteins are necessary for immunisation, screening, and analytical method development. However, by leveraging virus-like particles, KACTUS successfully produced full-length Claudin 18.2 protein in mammalian cells. Their consistent activity and purity testing results demonstrate the functional integrity and bioactivity of their VLP-displayed protein platform.

Dynamic Light Scattering Data for KACTUS Claudin 18.2 virus-like particle (VLP) shows uniform size of the VLP membrane proteins — Figure 1. This graph illustrates the intensity distribution of Claudin 18.2 VLPs as measured by dynamic light scattering (DLS). The peak centered at approximately 150 nm indicates a homogeneous population of VLPs, reflecting the consistent size and quality of the particles.

HPLC Data for KACTUS Claudin 18.2 virus-like particle (VLP) demonstrates purity of full length VLP membrane proteins for antibody drug development — Figure 2. The chromatogram shows the high-performance liquid chromatography (HPLC) profile of Claudin 18.2 VLPs, with a prominent peak observed at 4.909 minutes, indicating the retention time of the Claudin 18.2 protein. The sharp and well-defined peak suggests a high purity and consistency of the VLP preparation.

ELISA data for Claudin 18.2 virus-like particle (VLP) — Figure 3. ELISA assay shows the binding activity of Claudin 18.2 VLPs to anti-Claudin 18.2 antibodies across three different production batches. The consistent curves for Batches 1, 2, and 3 highlight the reproducibility and stability of the VLP preparations. The half-maximal effective concentration (EC50) is determined to be 0.1 nM, indicating high affinity and robust interaction between Claudin 18.2 VLPs and the specific antibodies.

BLI data for KACTUS Claudin 18.2 virus-like particle (VLP) validates high binding affinity of full length membrane protein VLP for antibody development — Figure 4. Biolayer Interferometry (BLI) data demonstrates the binding kinetics of Claudin 18.2 VLPs to streptavidin-labeled probes. The analysis reveals a high binding affinity with a dissociation constant (KD) of approximately 5.73E-10 M, indicating strong and stable interaction.

FACS data for KACTUS Claudin 18.2 virus-like particle (VLP) validates flow cytometry bioactivity performance of full length membrane protein VLPs — Figure 5. Flow cytometry data demonstrates the binding specificity and quantification of Claudin 18.2 displayed on VLPs. The top panels show the results from HEK293 control cells, while the bottom panels show the results from anti-Cld18.2 CAR-expressing HEK293 cells. The presence of Cld18.2 VLPs results in a significant shift in the fluorescence signal in anti-Cld18.2 CAR-expressing cells, indicating successful detection and quantification of Cld18.2 on VLPs. This validates the high-quality expression and bioactivity of the Claudin 18.2 VLPs.

SPR data for KACTUS Claudin 18.2 virus-like particle (VLP) validates bioactivity performance of full length membrane protein VLPs for antibody development — Figure 6. SPR data showing the binding interactions of Biotinylated Claudin 18.2 VLPs with streptavidin immobilised on a CM5 chip. Results show a dissociation constant (KD) of 1.638E-11 M. This indicates a very strong and stable binding affinity of the Claudin 18.2 VLP.

Nanodisc Proteins

Nanodiscs are small disc-shaped structures that mimic a cell membrane. A target protein is present in a lipid bilayer, which is surrounded by membrane scaffold proteins or copolymers. This discoidal structure enables solubilisation without the use of detergents. At KACTUS, they use a proprietary amphipathic copolymer to ensure performance and reliability. Their nanodiscs can be applied in applications such as ELISA, SPR, BLI, and yeast display.

Diagram showing expression of KACTUS copolymer nanodiscs with detergent free extraction and full-length transmembrane proteins. — *Schematic diagram of nanodisc displaying a multi-transmembrane protein*

Advantages of Nanodisc Proteins

Using nanodisc-displayed membrane proteins has several benefits. It maintains the natural conformation of the protein, provides water-soluble proteins, and displays the protein in a native-like environment. Moreover, because nanodiscs can be extracted without detergents, they are superior for membrane proteins that are sensitive to maintaining activity with detergents.

Applications

Yeast display screening
Functional in vitro assays
CAR expression testing
PK/PD Studies
Analytical Tests, ELISA, SPR, BLI

Features

Native protein structure and conformation
Detergent-free extraction
Water-soluble
Proven biological activity via ELISA and SPR
Mammalian cell expression system for natural folding and glycosylation

Product Validation Data

GPRC5D Nanodisc

GPRC5D is a complex, 7-transmembrane domain protein and a popular target for multiple myeloma treatment. This protein is being targeted in CAR-T cell therapy, antibody-drug conjugates, and bispecific antibody drugs (with CD3 protein) for myeloma. To facilitate GPRC5D drug development, KACTUS has developed a full-length GPRC5D nanodisc with verified bioactivity via ELISA and SPR. Along these lines, they have developed biotinylated nanodisc proteins to broaden use cases for this membrane protein. Their biotinylated GPRC5D nanodisc has high binding activity and can be used in analytical method development and pharmacokinetic analysis.

ELISA data of KACTUS GPRC5D copolymer nanodisc bioactivity of full length membrane protein nanodisc. — Figure 7. Human GPRC5D Nanodisc with His Tag was immobilised at 2 µg/ml (100 µl/well) on the plate. The dose-response curve for the Anti-GPRC5D Antibody with hFc Tag was generated, with an EC50 of 4.9 ng/ml as determined by ELISA.

ELISA data of KACTUS GPRC5D copolymer nanodisc verifies bioactivity of full length membrane protein nanodisc — Figure 8. Human CD3E&CD3D, with a His Tag, was immobilised on the plate at a concentration of 2 μg/mL (100 μL/well). Serial dilutions of the Anti-Human CD3×GPRC5D bispecific antibody, with an hFc Tag, were then added, followed by the addition of biotinylated Human GPRC5D Nanodisc, with a His Tag, at 5 μg/mL. Detection was performed using HRP-conjugated streptavidin, and the EC50 was determined to be 0.28 μg/mL by ELISA.

BLI data of KACTUS GPRC5D copolymer nanodisc verifies bioactivity performance of full length membrane protein nanodisc — Figure 9. Biotinylated Human GPRC5D Nanodisc, with a His Tag, was loaded onto an SA-Biosensor. This setup was used to determine the binding affinity to the Anti-GPRC5D Antibody, which was measured to be 1.16 nM using a BLI assay.

SPR data of KACTUS GPRC5D copolymer nanodisc verified bioactivity performance of full length membrane protein nanodisc — Figure 10. Human GPRC5D Nanodisc with His Tag was captured on a CM5 Chip via an anti-His antibody. It was found to bind the Anti-GPRC5D Antibody with an affinity constant of 1.47 nM, as determined by SPR assay (Biacore T200).

Caltag Medsystems is the distributor of KACTUS products in the UK and Ireland. If you have any questions about these products, please contact us.

KACTUS: Transmembrane Proteins