Backed by its recognized and experienced scientific team, CALIXAR provides a wide range of services in the field of membrane proteins. The following picture represents the different activities that are conducted at CALIXAR.
The main part of our activities is membrane protein production. Expression trials are performed using different expression systems (described below) with different constructions of the protein of interest. We select the best systems and constructions that respect the quality, the quantity and the integrity of the protein of interest. This selection is done by applying different extraction, stabilization and purification steps. During this process, we control by specific assays that the protein maintains its structural and functional integrity (see below).
Our discovery platform also allows deorphanization activity, identification and localization work. We can indeed search for the corresponding ligand of a given membrane protein or identify the corresponding membrane protein of a given ligand. We are also able to identify natural membrane protein partners using our approach of native solubilization / enrichment combined with mass spectrometry (Rosati, A., et al., 2015, Nature Communication). In addition to that, we can also look for membrane protein localization (plasma or internal membranes) by applying specific fractionation methodologies.
Starting materials can be either endogenous or recombinant system.
Endogenous systems include organs, virus, bacteria and primary cells. The idea with these specific systems is to extract and enrich membrane proteins from different biological membranes while maintaining their structural and functional integrities. As an example, these endogenous systems can be used to identify natural membrane protein partners if combined to mass spectrometry. Therefore, the goal can be to better understand the mechanism of action in most native state. Some case studies highlight our approach starting from brain, heart, inactivated viruses, pathogenic bacteria and primary cells (macrophages, dendritic cells or cancer cells).
We are using different recombinant systems in our laboratory including E.coli, Yeast, HEK cells, CHO cells and insect cells (baculovirus) to overexpress membrane proteins of interest. BacMam expression system is typically used to infect HEK and CHO cells. Since there is no perfect system for membrane proteins, we are open to test and implement different systems more adapted to the nature of the protein and to the application.
How to qualify your expression system ?
During the membrane protein production process, we evaluate the quality of the protein to ensure that its structural and functional integrities are maintained. Different tests are performed as follows.
Tests can be adapted depending on the type of protein and the applications. As for standard controls we are routinely using techniques such as Western blots, Coomassie gels and Silver stain among others. We also use Size Exclusion chromatography (SEC) and Native PAGE to confirm that the protein is not aggregated. Then we evaluate the thermostability or the stability overtime in addition to freeze/ thaw cycles.
Regarding the functionality of the protein, we first need to look out the family of protein which is investigated. For GPCRs, we test the binding capabilities using radioligand binding, SPR and/or NMR (Igonet S et al., 2018, Scientific report). For enzymatic proteins, we do enzymatic assays such as ATPase assays. For ion channels, we can look into toxin binding and/or current voltage measurement in presence and absence of specific inhibitors (Agez M et al., 2017, Scientific reports). We also perform ELISA when protein partners or antibodies are available. If no assays are available and if there is a need to develop one, CALIXAR can propose to design one. Otherwise, the most important Q/C is to avoid aggregates and have homogenous populations of the purified proteins. This is determinant for the outcome of drug discovery.
We have developed and reported a systematic solubilization approach based on the use of Dot Blot as readout (Mandon E et al., 2016, Analytical Biochem). The idea in this specific experiment is to biotinylate lipids and then use different solubilization conditions (proprietary compounds alone or in combination with commercially available detergents) in a 96 well plate format, to solubilize membrane proteins and then use streptavidin beads to deplete the preparation on membranes that were not solubilized.
Everything which is solubilized will be in the supernatant and everything which is not solubilized (membranes) will be removed from the supernatant thanks to this streptavidin beads. The supernatant is then analyzed by Dot Blot using specific antibody against the membrane protein of interest. We observe different intensities of signal and this intensity reflect to a certain extent the solubilization efficiency. Of course, to avoid false positives this is systematically further validated by Western Blot where we evaluate the molecular weight of the protein.
Above we can see what kind of results we can get from this kind of systematic solubilization. We used proprietary compounds (Calixarene-based or not) alone and combined with commercially available molecules (DDM, FC12, OG…). Some conditions give better solubilization efficiency than others. No detergent condition and SDS serve as negative and positive controls, respectively.