DiMPro^TM Full Length Transmembrane Proteins

Membrane proteins (MPs) are the gatekeepers of cells and play an important role in a variety of cellular functions such as material transport, signal transduction, and cell-to-cell recognition (Figure 1). Abnormal functions of MPs often lead to the occurrence of diseases. Therefore, MPs account for more than 60% of all the current FDA-approved drug targets and 90% of the antibody drug targets.

Figure 1: Membrane proteins play important roles in a variety of cellular functions.

Despite the importance, preparation of functional proteins for drug discovery is challenging, especially for multi-pass transmembrane proteins. Membrane proteins tend to lose the functions when removed from the membrane. The expression level of membrane protein is usually low in the host cells. Cellular expression of recombinant membrane protein often results in protein aggregation and misfolding due to the hydrophobic nature of transmembrane segments. Therefore, membrane proteins have been considered as “undruggable” targets in pharmaceutical companies.

Antigen Preparation

To date, various antigens have been used for the preparation of mAbs against membrane protein (MP). Each of them has shown its own benefits and issues (Table 1). Extracellular domains (ECDs) are easy to be purified at high expression level. But they normally don’t have native conformation of target proteins, which can cause the loss of protein activities and some conformational epitopes. Whole cell immunization has been widely used for the mAb generation against transmembrane proteins. It maintains the native conformation and modifications of target proteins, has high immunogenicity and doesn’t require complex purification steps. However, whole cell immunization often generates a lot of non-specific antibodies against other proteins, especially the abundant cytoplasmic proteins.
To solve this issue, DIMA Biotech has developed several platforms for the full-length membrane proteins (MPs), including Nanodisc, Virus-like particles (VLPs), Membrane Nanoparticles (MNPs), and exosome (EXO) et al. (Figure 2). All platforms are based on mammalian expression systems and prepare full-length membrane proteins in soluble protein products with native conformation and activity. They can be used for the development of highly specific therapies targeting previously intractable targets such as ion channels and transporter.

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Figure 2: DIMA’s solutions for the full-length multi-pass transmembrane proteins

Table 1: Benefits and issues of different antigens

Virus-like particles (VLPs)

Virus-like particles (VLPs) are self-assembling multi-protein nanoparticles with similar structural organization and conformation as viruses but without viral genome. The size of the VLP is about 100-150nm. It is secreted from the surface of the cells that express target membrane proteins (MPs). The purified VLPs have the target MPs inserted in a complete bilayer phospholipid membrane structure, mimic the natural membrane-penetrating state of the protein (Figure 3).
VLPs can be used for routine biochemical analysis, including ELISA, SPR affinity analysis, phage display screenings, protein labeling and cell binding experiments, Flow virometry analysis, etc. It can also be used as functional protein antigens to develop active antibodies with high drug potentials because the target protein on VLP exhibits a state like its native state on the cell surface.

Figure 3: Virus-like particle (VLP) for the preparation of the full-length multi-pass transmembrane proteins

Featured VLPs Products

Human Claudin-18.1 full length protein-VLP
Human Claudin-18.2 full length protein-VLP
Human CCR8 full length protein-VLP
Human CCR4 full length protein-VLP
Human CD24 full length protein-VLP
Human GPRC5D full length protein-VLP

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Figure 4: ELISA plates were pre-coated with 0.5ug/per well purified human Claudin 18.2 VLP. Serial diluted Anti-Claudin18.2 monoclonal antibody (Zolbetuximab biosililar; IMAB362) solutions were added, washed, and incubated with secondary antibody before ELISA reading. From above data, the EC50 for IMAB362 binding with Claudin18.2 is 15.37ng/ml.

Figure 5: Flow analysis of Claudin18.2 VLP

Claudin18.2 VLP samples were stained only with Goat anti-human IgG Fc-PE secondary antibody.
Control VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Claudin18.2 VLP samples were stained with an irrelevant antibody at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Claudin18.2 VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.

Synthetic Nanodisc

Unlike other membrane scaffold protein (MSP) Nanodisc on the market, synthetic Nanodisc developed by DIMA Bio can be prepared directly from the cells. The polymers used during this process have a dual function. It dissolves the cell membranes, like the detergent, and uses cellular phospholipids to form Nanodisc around the membrane proteins. The target protein embedded Nanodiscs can then be purified (Figure 6).

Figure 6: Synthetic Nanodisc for the preparation of the full-length multipass transmembrane proteins

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Membrane Nanoparticles (MNPs)

Plasma membrane-coated nanoparticles (MNPs) have been used in various applications, including delivery of therapeutic agents and induction of immune responses et al. Unlike the conventional strategies, MNPs directly leverage intact and natural functions of cell membranes, and show high biocompatibility, specificity, and low side effects. DIMA biotech developed an optimized MNPs platform for the full-length membrane protein productions using membrane coating technology and HEK293 based expression platform. The high-purity plasma membrane-coated nanoparticles were produced by extrusion after membrane extraction from the host HEK293 cells containing the overexpressed target proteins (Figure 7).

Figure 7: Membrane Nanoparticles (MNP) for the preparation of the full-length multi-pass transmembrane proteins

Featured MNP Products

Human CLDN6 full length protein-MNP
Human CCR8 full length protein-MNP
Human CD24 full length protein-MNP

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Figure 8: Western blot of CCR8 membrane nanoparticles

Figure 9: Flow cytometry validation of CCR8 MNP protein

A. CCR8 full length membrane nanoparticles samples were stained only with Goat anti-human lgG 488 secondary antibody.
B. Control membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
C. CCR8 full length membrane nanoparticles samples were stained with an irrelevant antibody at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
D. CCR8 full length membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.

Exosomes (EXO)

Exosomes (EXO) are secreted membrane nanoparticles formed by fusion of multivesicular bodies (MVBs) with the plasma membrane. The size of exosomes is about 30-150nm. Exosomes can be purified from in vitro cultures of cells. Like VLPs, EXO also shows outstanding immunogenicity and have the membrane structure close to that of the native plasma membrane. Overexpressed membrane proteins (MPs) were inserted in the host cell membrane and secreted out as Exosomes which can be purified and used for a series of downstream studies. Unlike VLPs, the purified EXO has minimal cytotoxicity. However, not all MPs can be included in EXO and secreted into medium.

Figure 10: Exosomes (EXO) for the preparation of the full-length multipass transmembrane proteins

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Figure 11. ELISA plates were pre-coated with 0.5 μg/per well purified human CD24 exosome. Serial diluted Anti-CD24 monoclonal antibody solutions were added, washed, and incubated with secondary antibody before ELISA reading. From above data, the EC50 is 69.61 ng/ml.

Figure 12. Nanoparticle Tracking Analysis of CD24 exosomes

Figure 13. TEM image of CD24 exosomes

Detergent Solubilization of Membrane Proteins

Detergents are amphipathic molecules containing hydrophilic polar heads and non-polar hydrophobic tails. At certain concentration, detergent can form stable micelles with hydrophobic cores which can be used to isolate the active membrane proteins (MPs) in soluble protein-detergent complexes. Figure 6 demonstrates how to use detergent to isolate the full-length multipass transmembrane proteins, and to maintain the hydrophobic transmembrane structure of the membrane proteins.

Detergent for full length membrane protein purification

Figure 14: Detergent for the preparation of the full-length multipass transmembrane proteins

DiMProTM Full Length Transmembrane Proteins