Poster Open Access

Stabilization of native & functional membrane proteins: CALIXAR approach

Elodie Mandon

Membrane proteins represents up to 70% of therapeutic targets that are involved in different key cellular processes and signaling events. Surprisingly, less than 1% of protein structures in the Protein Data Bank correspond to membrane proteins. Those targets are very often instable which represents a bottleneck for their production in homogenous and stable state suitable for structural studies, antibody development and HTS ligand screening. CALIXAR has developed an innovative approach consisting on native isolation of therapeutic targets from the expression, solubilization/stabilisation, and purification to functional as well as structural characterization. Instead of modifying the protein (by truncations and mutations) to adapt to the environment, we modify the chemical environment to adapt to the protein, using innovative proprietary detergents/ surfactants compounds and combination of compounds to help solubilize and stabilize in the same time. The starting material can be either endogenous (Organs, Primary cells, bacteria, and viruses), or recombinant (E.coli, Yeast, insect cells, CHO and HEK cells). Here we will describe examples of highly druggable targets produced in their most native state without any single mutation, truncation or fusion and that could exhibit striking thermo-stability increase and homogeneity improvement, while keeping their functional features. Indeed, more than 30C thermostability improvement was obtained for the human transporter BCRP while keeping its native sequence and modifying only the chemical environment. We show striking stability improvement of 7TM proteins: bacteriorhodopsin and wild type and non-truncated adenosine receptor for which crystals could be obtained and antibodies were derived. Finally, conditions of functional stabilization of the trimer of HIV envelop protein were also found. This will pave the way to future structural investigations and conformational antibody development of native HIV envelop.

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