Supporting innovative drug development with full-length transmembrane protein platforms

transmembrane protein platforms, drug development

An essential step in preclinical drug development is confirming the specific binding of the novel therapeutic molecule to the protein target. Transmembrane proteins (TPs) such as ion channels, receptors, or transporters present a large group of targets involved in cancer and other diseases. Due to their complexity, producing those proteins in sufficient amounts is far more challenging than for their soluble counterparts, presenting a bottleneck in the drug discovery workflow.

Novel approaches to facilitate drug development include full-length transmembrane protein platforms, offering purified, high-quality TPs as off-the-shelf items for use in drug-target interaction analyses.

Meet the Claudin family: Gatekeepers in cancer

Examples of TP drug targets can be found in the Claudin protein family, a group of TPs playing a central role in the tight junctions – gatekeeping structures that regulate the flow of ions and water between epithelial and endothelial cells. 

Among other functions, tight junctions support tissue structure integrity, preventing the migration of cancer cells through the endothelial barrier into the bloodstream. Recent studies have shown that abnormal expression rates of Claudin protein family members are related to tumor growth and metastasis, making them relevant therapeutic targets and cancer prognosis factors. 

Claudins consist of four transmembrane-spanning domains with peptide loops located on the outside of the membrane. Together, those loops form three-dimensional structures or epitopes targeted by therapeutic or diagnostic antibodies.  

Spencer Chiang, Communication Manager at ACROBiosystems
Dr. Spencer Chiang, Communication Manager at ACROBiosystems

“Producing only the isolated soluble loops of the Claudin proteins would be fast and easy. However, we know that because the individual peptides do not interact correctly, the three-dimensional structure required for reliable antibody screening does not form. But when we express Claudins as full-length TPs, the transmembrane domains hold the external loops in the correct position,” explained Dr. Spencer Chiang, Communication Manager at ACROBiosystems. 

Interaction studies with therapeutic molecules and antibodies can only yield meaningful results when the target is presented in its biologically relevant conformation, calling for methods that enable the production of correctly folded, active, full-length TPs. Those methods, however, are still challenging to establish and vary between individual protein targets.

Overcoming the challenges of transmembrane protein production

But why is it so difficult to prepare high-quality, full-length TPs compared to their soluble counterparts?

First, TPs tend to be rather large, and there is minimal free surface area on the host cell membranes, which TPs need to be expressed in their native conformation.

TPs that cannot fold correctly because of a lack of membrane space tend to aggregate, as their “water-avoiding” (hydrophobic) regions stick together, forming inactive protein clusters. Resolubilizing and refolding aggregated TPs to an active state is tricky and often unsuccessful, affecting the quality of the TP preparation.

Those TPs that successfully insert into the host cell membrane may be functional – maintaining their native function, interfering with the careful metabolic balance, often in a harmful or even toxic way. Expression of foreign TPs, therefore, needs to be optimized to protect the host cell and prevent cellular toxicity.

 “Our experts at ACROBiosystems have carefully optimized each step of the manufacturing process – from expression vector construction to expression and purification – to obtain sufficient yields without compromising quality, creating the Full-Length Active Gallery (FLAG) Platform. A crucial part of the FLAG approach is that we confirm activity for each of our products using several established methods to ensure suitability for our clients’ applications,” stated Chiang. 

transmembrane protein platforms, drug development

Tailoring stabilization formats to drug development analysis methods 

Because TPs are not stable outside of a lipid or lipid-like environment that shields the hydrophobic protein domains, stabilizing agents are necessary. ACROBiosystems uses three different approaches for transmembrane protein stabilization, tailored to their clients’ planned applications and research questions. 

The most common option is to produce the TPs in insect or mammalian cell cultures and isolate them using detergents that form a micelle around the hydrophobic area, keeping the protein from aggregating. Detergents have been used for decades, and a broad range of chemical entities is available. The ACROBiosystems team will choose the best one for the individual TP and optimize its formulation. 

“One of the biggest advantages of detergent-stabilized TPs is their ability to be precisely quantified. They are suitable for many in vitro assays such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR),” explained Chiang. “At the same time, we know that many cell-based assays are not compatible with detergents.”

A detergent-free alternative is the use of virus-like particles (VLPs) that transfect mammalian cell cultures, so they produce the transmembrane protein and give rise to budding virus-like particles displaying the transmembrane protein on their surface. VLPs provide a large membrane surface area disconnected from cell metabolism, permitting higher expression rates.

VLPs are suitable for immunization projects and cell-based assays, e.g., detecting chimeric antigen receptors (CARs). A recent case study demonstrated the specific binding of Claudin 18.2-VLP to Claudin-18.2.-CAR-293 cells.

transmembrane protein platforms, drug development

Expanding the FLAG platform: Nanodiscs, Claudins, GPCRs, and beyond

Nanodisc technology is one of the most recent developments in TP stabilization. Nanodiscs are synthetic lipid bilayer structures composed of membrane scaffold proteins and phospholipid molecules. TPs can be integrated within the nanodisc structure while maintaining their native conformation and activity in a detergent-free environment. 

“Nanodiscs not only stabilize the transmembrane protein but also allow studies of the protein area at the inside of the cell membrane, which is normally hidden,” stated Chiang. 

Currently, ACROBiosystems offers almost 50 off-the-shelf items, including four Claudin family members, several immune receptors, G-protein coupled receptors (GPCRs), and corresponding control proteins to help exclude non-specific interactions.

While the catalog of TPs is constantly growing, customized projects on new protein targets are always welcome. The next steps will include extending the Claudin protein family and adding more GPCRs to the offering – TPs relevant, for example, to cancer, metabolic diseases, and neurobiology. 

“We are happy that with our comprehensive FLAG transmembrane protein and technology platform, we can support the development of innovative therapies for cancer and other serious conditions,” concluded Chiang.

Learn more about the FLAG transmembrane protein and technology platform at ACROBiosystems.

Explore other topics: CancerDrug developmentprotein

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