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  • Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

Scientific reports (2019-09-25)
Morgane Agez, Elodie Desuzinges Mandon, Thomas Iwema, Reto Gianotti, Florian Limani, Sylvia Herter, Ekkehard Mössner, Eric A Kusznir, Sylwia Huber, Matthias Lauer, Philippe Ringler, Claudia Ferrara, Christian Klein, Anass Jawhari
ABSTRACT

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

MATERIALI
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Sigma-Aldrich
Cocktail di inibitori delle proteasi in compresse, senza EDTA SIGMAFAST, for use in purification of Histidine-tagged proteins
Sigma-Aldrich
Anti-polyHistidine−Peroxidase antibody, Mouse monoclonal, clone HIS-1, purified from hybridoma cell culture
Millipore
SNAP i.d. 2.0 Protein Detection System-Mini and MultiBlot (7.5 x 8.4 cm and 4.5 x 8.4 cm), Developed to meet the needs of our Western blotting customers, the SNAP i.d. 2.0 system produces blots of a very high quality. Unique vacuum-driven technology & a built-in flow distributor actively drive reagents through the membrane.