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Ion Channel Preparation as Immunogen

Creative Biolabs established a Magic™ membrane protein platform. With experienced scientific team and leading technologies, now we expand new service to prepare ion channel protein as immunogen for antibody studies.

Integral proteins that function in the transport of ions, small molecules and macromolecules across biological membrane are called membrane transport proteins. Channel protein is one of the two basic types of membrane transport proteins (Fig 1). Ion channel proteins are ion selective and contain a water-filled tunnel to facilitate the diffusion of ions and water molecules across biological membranes quickly when the channel is open in response to given stimulus. There are mainly voltage-gated channels (Ca2+ channel, Na+ channel, K+ channel, Cl- channel and H+ channel proteins) and ligand-gated channels (ionotropic glutamate-gated receptors, GABAA receptor, etc). In addition, ion channels have complex topologies.

Illustration of channel proteins.Fig. 1 Illustration of channel proteins. (Marbán E.2002)

Ion channels participate in establishing resting membrane potentials, regulating cell volume, changing action potentials and transmitting electrical signals by the flow of ions across cell membrane. As a result of their multiple functions, ion channels are playing critical roles in many biological processes, and manifest as an increasingly attractive therapeutic target, which comprises the third best-selling group of prescribed drugs all over the world. However, the study of ion channels is greatly hindered by their complicated structure, low expression abundance, and difficulty in maintaining intact functional properties. Based on years of exploration and non-stop efforts, scientists in Creative Biolabs has developed a featured membrane protein technique platform, which successfully solved many challenging membrane protein targets, including 12 TM, 20 TM ion channels. We now offer professional service of ion channel preparation as immunogens using following strategies:


Proteoliposomes are a popular immunogen type and widely applied in many programs. Highly purified ion channel proteins inserted into liposomes can well maintain their native structure and activities. Proteoliposomes can be prepared from either cell-based expression or cell-free expression systems. With the aid of our Lipid Screening Platform, we will perform comprehensive condition screening to find the optimal lipid components. Our scientists have vast experience in raising high specific, high-affinity binders against membrane proteins in liposome format.


Nanodiscs make it possible to fix the transmembrane region of ion channels in a native-like phospholipid bilayer environment. It offers a convenient reconstitution format not only highly mimics the natural plasma membrane, but also very flexible in terms of size and composition. Besides, in comparison to liposomes or micelles, ion channel nanodiscs are also more stable. As immunogens, nanodiscs can ensure minimal immune response against the transmembrane region, thus raise more homogeneous antibodies targeting excellular/intracellular domains. We offer high-quality nanodisc assemble service from both cell-based expression and cell-free expression systems.


Polymers, including amphipols and SMALPs (or co-polymers), emerge as a novel approach for ion channel reconstitution in recent years. Unlike detergent, it does not have to disrupt the native lipid environment around the target protein; instead, it can directly isolate and solubilize membrane-lipid complex. This detergent-free method could prepare immunogen with maximally preserved structure and activities.


In this approach, cells are co-transfected with vectors encoding target ion channels and viral core proteins (e.g. Gag). After budding off from host cells, the viral core will capture plasma membrane fractions enriched with the target protein, also termed as lipoparticles. The lipoparticles displaying the target can then be used for a wide variety of applications, such as animal immunization, antibody screening, drug delivery, assay development, etc.


Peptide/protein derived from extracellular/intracellular domains is also a common-used format for integral membrane proteins, particularly for ion channels. Since they commonly have large extracellular/intracellular loops or regions. To our knowledge, the extracellular and intracellular loops are the primary functional regions and play key roles in interacting with ligands or substrates. Hence, carefully designed and characterized surrogate proteins/peptides can serve as good immunogens and raise many potent binders.


Apart from all the complicated preparation procedures for recombinant ion channels, another strategy is to directly use overexpressing whole cells for immunization as well as antibody selection. This method is frequently used for anti-membrane protein antibody discovery, which has proved remarkable success in many cases. Several key points for whole cell immunogen include sufficient expression levels, appropriate immunization process, and adequate antibody screening. Currently, we have well-established stable cell lines (HEK293/CHO/U2OS background) of over 100 GPCR/ ion channels. Customized stable cell line can also be constructed upon request.


Exosomes are secreted membrane nanovesicles (diameter 50-100 nm) that form within late endosomal compartments by the fusion of multivesicular bodies with the plasma membrane. Due to the formation mechanism, membrane orientation of exosomes is close to the orientation of the whole cell. Exosomes secreted from dendritic cells share some similar properties of dendritic cells. Incorporating ion channel in exosomes could cause strong immune responses without additional adjuvant in animals when used as immunogen. Besides, exosomes provide a native membrane environment and have stable storage property.

With numerous successful cases, scientists in Creative Biolabs have confidence to assist our clients to design and prepare the best ion channel immunogens for their antibody development projects. Meanwhile, we provide one-stop professional pipeline to the downstream antibody selection and purification ranging from small to large scales. Please feel free to contact us for more details.

Reference

  1. Marbán E. (2002). Cardiac channelopathies. Nature, 415(6868), 213-218.



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