Creative Biolabs has cooperated a novel technology platform, Magic™ conversable mammalian library (CML), combing the mammalian-cell expression library and cell surface display library. This combined system not only improves the successful expression of prey proteins but also facilitates cell-based screening for protein-protein interactions.
Magic™ Conversable Mammalian Library (CML) Platform
In the field of new therapeutics development, target-based and phenotypic-based drug discovery are the two fundamental strategies. The latter strategy is based on the exploration and characterization of drug molecules by identification interacting proteins within tissues and cells and elucidating the mode of actions. There have been various methods used for novel interactions discovery, such as Y2H, B2H, M2H etc., which are based on the library with a soluble- or cell surface display- pattern.
The novel conversable library combines the two patterns. As shown in the Fig.1, each prey protein is constructed to fuse with a Fab, the Fab facilitates the conversion between different library formats. Each prey-Fab fusion protein fuses with a purification tag for soluble protein expression and a transmembrane (TM) domain for cell surface display. A classical application using this platform is to discover the pairing of receptors.
Fig.1 Schematic of the conversable mammalian library (Yang, 2017)
The Pairing of Receptors using Magic™ CML Platform
Interactions between protein ligands and receptors play an important role in cell-cell communication and signal intermediate. Orphan receptor refers to a protein that has a structural similarity to other known receptors but its endogenous ligand or activators has not been identified yet. Likewise, there are also some ligands whose cognate receptor is unknown. The Magic™ CML platform is an appropriate option to solve this problem.
Fig.2 Hepatocyte nuclear factor alpha (HNF4-a): a mammalian orphan nuclear receptor (ONR). (Gallastegui, 2015)
We can provide one-stop discovery services for the pairing of receptors using the Magic™ CML platform. The workflow is as shown in the Fig.3. In this strategy, the extracellular domains (ECDs) of receptors is constructed as a Fab-fusion protein. Each ECD-Fab fusion protein contains two constructs: one is the fusion protein linked to a purification tag for soluble protein expression and the other one is that linked to a transmembrane (TM) domain for cell surface anchored and display. The two constructs are transfected into mammalian cells to construct the conversable library. For soluble proteins expression, the supernatant and purified products can be used for the further high throughput screening and functional assays. While the protein-anchored cells can be analyzed by FACS or other cell-based screening assays.
Fig.3 Workflow of the expression library production and primary screening. (Yang, 2017)
The Magic™ CML platform combines the advantages of soluble proteins screening and cell surface display technology to discover novel interactions. If you’re interested in the platform or have any question for other biomolecular interaction technologies, please feel free to contact us for more details.