Transmembrane Proteins

Synthetic Nanodiscs

Nanodiscs are nanoscale structures (10-50 nm) comprising transmembrane proteins within a phospholipid bilayer, held by a stabilising belt. First conceived by the laboratory of Professor Stephen G. Sligar in 2002³, nanodiscs offer a stable and biologically relevant environment to enable the isolation of TMPs in their native conformation.

Synthetic nanodiscs, one of the two types of nanodisc, are composed of native cell membrane phospholipids encircled by belts made from a variety of synthetic polymers, such as styrene-maleic acid co-polymers (SMAs) or Diisobutylene-maleic acid (DIBMA), offering great control and versatility in application^4,5. These synthetic belts have been engineered for stability and solubility, meaning no detergents are required in the isolation of transmembrane proteins held within the nanodiscs. Furthermore, nanodisc proteins are produced in mammalian cell expression systems, which ensure proper folding and post-translational modifications (PTMs) of proteins. This is particularly important for complex multi-pass transmembrane proteins, which often require specific chaperones and co-factors for assembly.

Figure 2: Synthetic nanodiscs containing full-length TMPs in a phospholipid bilayer are generated from native cell membranes.

Advantages of Nanodiscs:

• Detergent-free purification process.
• They provide a stable environment for TMPs in vitro - preserving their natural conformation and activity.
• They are versatile and can be employed across numerous applications (see below).
• Nanodiscs show promise in vaccine preparation and antibody production.

Nanodisc Applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)
• Nanodiscs facilitate immobilisation on ELISA plates for detection.
• Surface Plasmon Resonance (SPR) Affinity Analysis
• Through immobilisation of transmembrane proteins on SPR chips.
• Chimeric Antigen Receptor (CAR) Expression Analysis
• Incorporation of CARs into nanodiscs for downstream characterization.
• Protein Crystallisation
• Enables TMPs to be stabilised and subjected to crystallisation conditions for structural analysis.
• Phage Display Screening
• Transmembrane proteins within nanodiscs can be targeted in phage display libraries.
• Toxicology
• To study the effects of drugs or environmental factors on transmembrane proteins.
• Vaccine Development
• Enhance stability and immunogenicity of transmembrane protein antigens.

Figure 3: Synthetic Nanodisc Human TLR4 Protein (A318415) can bind Anti-Claudin18.2 Chimeric Antibody [Zolbetuximab Biosimilar] - Azide free (A318887) with an affinity constant of 1.619 nM as determined in a SPR assay.

Figure 4: Immobilized Synthetic Nanodisc Human CCR8 Protein (A318461) binds Anti-CCR8 Humanized Antibody [BMS 986340] - Azide free (A318852). The EC50 for this binding is 12.07 µg/ml.

Virus-like Particles (VLPs)

Virus-like particles (VLPs) are non-infectious structures that mimic the shape of viruses but lack a viral genome. During VLP production in cells or cell-free systems, VLPs self-assemble into 100-150 nm protein particles, and incorporate transmembrane proteins into a VLP-MP complex, resulting in presentation of the protein on the external surface of the VLP.

Figure 5: VLP-TMP complexes are generated containing full-length transmembrane proteins following self-assembly.

Advantages of VLPs:

• Proteins are highly expressed and purified without the need for detergents.
• VLP-TMPs deliver high quality immunogens for antibody development and screening.
• The transmembrane protein is maintained in its native structure and orientation.
• Engineered to have only specific transmembrane protein molecules on VLP surface.
• High immunogenicity for vaccine production.

VLP Applications:

• Vaccine Development
• VLPs can be based on a variety of viruses, including hepatitis B virus, human papillomavirus and influenza virus.
• Surface Plasmon Resonance (SPR) Affinity Analysis
• Through immobilisation of transmembrane proteins on SPR chips.
• Antibody Production
• VLPs are useful for displaying transmembrane proteins at high density.
• Ligand Binding Experiments
• Used to analyse kinetics of receptor-ligand binding.
• Drug Discovery
• The isolated VLP-TMP may be used to screen large compound libraries in a high throughput screening format.

Figure 6: Immobilized Synthetic Virus-like Particle Human Claudin 6 Protein (A318460) binds Anti-Claudin 6 Humanized Antibody [IMAB027] - Azide free (A318880). The EC50 for this binding is 5.616 µg/ml.

Figure 7: Immobilized Synthetic Virus-like Particle Human Claudin18.2 Protein (A318490) binds Anti-Claudin18.2 Chimeric Antibody [Zolbetuximab Biosimilar] - Azide free (A318887). The EC50 for this binding is 15.37 µg/ml.

Exosomes

Exosomes are small (30-150 nm) membrane-bound vesicles secreted by the cell, which mediate cell-cell communication and play an important role in the immune response. Each vesicle contains a variety of molecules including proteins, RNA and DNA.

Exosomes are useful for supporting transmembrane protein molecules by providing an optimal environment to maintain receptor solubility. The transmembrane proteins are overexpressed, localised to the host cell membrane, and subsequently secreted within exosomes, which are purified for downstream applications. As a result of the exosome being secreted, there is no need to perform harsh membrane extraction techniques which is sometimes necessary with traditional techniques.

Figure 8: Exosomes are naturally formed and secreted by cells, containing transmembrane proteins in the cell membrane as well as other biological molecules.

Advantages of Exosomes:

• High expression levels of the transmembrane protein.
• Detergent-free purification process.
• Mimics native structure and orientation of the natural transmembrane protein.
• Generates a strong immune response when used as an immunogen.

Exosome Applications:

• Antibody Development
• Due to high expression and concentration of the transmembrane protein in the exosome.
• Drug Discovery
• Screening compound libraries in an in vitro, high content screening assay.
• Characterising target TMPs
• Binding kinetics and biological function can be assayed when the MP is stably localised within exosomes.

Membrane Nanoparticles (MNPs)

Membrane nanoparticles (MNPs) comprise a synthetic nanoparticle coated with a layer of natural cell membrane. Membrane nanoparticles are extremely useful for isolating transmembrane proteins because of their small size (10-100 nm) and because the lipid bilayer maintains MP stability and solubility.

To form MNP-TMP complexes, the transmembrane protein is expressed within HEK293 cells and displayed on the cell surface, before proprietary extraction processes are used to isolate the membrane from the host cells and produce the target protein-containing membrane nanoparticles.

Figure 9: Overexpressed TMPs within the cell membrane are extracted and packaged as membrane nanoparticles.

Advantages of Membrane Nanoparticles (MNPs):

• Expression within HEK293 cells supports PTMs, and maintains native protein conformation and activity.
• Generate strong immune response when used as immunogens.
• Detergent-free purification process.
• Soluble in aqueous solutions for routine biochemical analysis.

Membrane Nanoparticles (MNPs) Applications:

• ELISA
• SPR Affinity Analysis
• Phage Display Screening
• Antibody Generation
• CAR T Cell Screening