DiMPro™ Full-length Transmembrane Proteins from DIMA Biotechnology

Original Article by DIMA Biotechnology

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

Despite their importance, the production of functional proteins for drug discovery is a challenge, especially for multi-transmembrane proteins. Membrane proteins tend to lose their functions when removed from the membrane and the expression level of membrane proteins is usually low in the host cells. Cellular expression of recombinant membrane proteins often leads to protein aggregation and misfolding due to the hydrophobic nature of the transmembrane segments. For this reason, membrane proteins are often considered "undruggable" targets in the pharmaceutical industry.

Our partner DIMA Biotechnology stands for innovation in the discovery of therapeutic antibodies and the expression of functional proteins. DIMA specialises in preclinical research and development solutions for the biopharma industry. The extensive portfolio comprises more than 1,200 antibodies and over 1,500 proteins and cytokines. This includes the full-length DiMPro™ transmembrane proteins, which are produced in mammalian expression systems as soluble protein products with native conformation and activity. In this blog article you will learn more about the different platforms DIMA has developed for the production of full-length human multi-transmembrane proteins.

These topics await you:

1) DIMA Platforms for the Production of human Multi-transmembrane Proteins

2) Virus-like Particles

3) Synthetic Nanodiscs

4) Membrane Nanoparticles

5) Exosomes

6) Detergent Solubilization of Membrane Proteins

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DIMA Platforms for the Production of human Multi-transmembrane Proteins

One of the most important prerequisites for the development of antibodies is the use of protein immunogens that best mimic the native state of the target protein. Most target proteins suitable for antibody-based therapy are membrane proteins, which are notoriously difficult to express. So far, different types of antigens have been used for the production of mAbs against MPs. Each of these types has its own advantages and disadvantages (Tab. 1).

Extracellular domains (ECDs) can be easily purified in high expression. However, they usually do not have the native conformation of the target proteins, which can lead to loss of protein activity and some conformational epitopes. Whole cell immunisation is often used for the production of mAb against transmembrane proteins. It preserves the native conformation and modifications of the target proteins, has a high immunogenicity and does not require complex purification steps. However, whole cell immunisation often generates many non-specific antibodies against other proteins, especially the abundant cytoplasmic proteins.

To address these issues, DIMA Biotech has developed five platforms for the production of full-length human multi-transmembrane proteins, including nanodiscs, virus-like particles (VLPs), membrane nanoparticles (MNPs) and exosomes (EXOs) (Fig. 1). All platforms are based on mammalian expression systems (HEK293) and produce membrane proteins as soluble protein products with native conformation and activity as well as authentic post-translational modifications. The use of serum-free medium also minimises the risk of contamination of host cells. The proteins produced can be used for the development of highly specific therapies that aim at previously inaccessible targets such as ion channels and transporters. Key applications of the proteins include:

1. Use as native immunogens for the development of therapeutic antibodies
2. Identification of native ligands and receptors
3. In vitro functional testing of therapeutic drugs
4. In vitro protein function assay
5. Cell-based assays

Figure 1: DIMA's solutions for the full-length multi-transmembrane proteins.  

To find the best production strategy, DIMA analyses and designs a separate expression construct for each target membrane protein. While for MPs with only one transmembrane domain they use the well-designed ECD fraction to mimic the functional domain, for full-length multi-transmembrane proteins, such as GPCR and claudin proteins, DIMA uses one of the five in-house developed platforms. These are characterised by new expression and extraction technologies and can be divided into two categories:

1. Native cell membranes, but impure proteins: membrane nanoparticles (MNPs), exosomes (EXOs) and virus-like particles (VLPs)
2. Pure proteins: detergent and nanodisc platforms

Virus-like Particles

Virus-like particles (VLPs) are self-assembling multiprotein nanoparticles with a similar structural organisation and conformation to viruses, but without a viral genome. The size of VLPs is about 100-150 nm. They are secreted from the surface of cells expressing the target membrane proteins. The purified VLPs have incorporated the target MP into a complete phospholipid membrane structure that mimics the natural membrane-penetrating state of the protein.

VLPs can be used for routine biochemical analyses, including ELISA, SPR affinity analyses, phage display screenings, protein labelling and cell binding experiments or flow virometry analyses. They can also be used as functional protein antigens to develop active antibodies with high drug potential, as the target protein in the VLP has a state that corresponds to its native state on the cell surface.

Synthetic Nanodiscs

In contrast to other membrane scaffold protein (MSP) nanodiscs on the market, the synthetic nanodiscs developed by DIMA Biotech can be produced directly from the cells. The polymers used in this process have a dual function. They dissolve the cell membranes, like the detergent, and utilise cellular phospholipids to form nanodiscs around the membrane proteins. The nanodiscs embedded in the target protein can then be purified.

Membrane Nanoparticles

Plasma membrane-coated nanoparticles (MNPs) have already been used for various applications, including the delivery of therapeutics and the triggering of immune responses. In contrast to conventional strategies, MNPs directly utilize the intact and natural functions of cell membranes and are characterized by high biocompatibility, specificity and low side effects. DIMA Biotech has developed an optimized MNP platform for the production of full-length membrane proteins using membrane coating technology and the HEK293-based expression platform. The high-purity plasma membrane-coated nanoparticles were produced by extrusion after membrane extraction from the HEK293 host cells containing the overexpressed target proteins.

Exosomes

Exosomes (EXOs) are secreted membrane nanoparticles that are formed by the fusion of multivesicular bodies with the plasma membrane. The size of exosomes is about 30-150 nm. Exosomes can be purified from in vitro cell cultures. Like VLP, EXOs also exhibit excellent immunogenicity and have a membrane structure close to that of the native plasma membrane. Overexpressed membrane proteins are inserted into the host cell membrane and excreted as exosomes, which can be purified and used for a number of downstream studies. In contrast to VLPs, the purified EXOs have minimal cytotoxicity. However, not all MPs can be taken up in EXOs and secreted into the medium.

Detergent Solubilization of Membrane Proteins

Detergents are amphipathic molecules with hydrophilic polar heads and non-polar hydrophobic tails. At a certain concentration, detergents can form stable micelles with hydrophobic cores that can be used to isolate the active membrane proteins in soluble protein-detergent complexes. The figure shows how detergents can be used to isolate full-length multi-transmembrane proteins and preserve the hydrophobic transmembrane structure of the membrane proteins.

All Full-length Proteins by DIMA Biotechnology