Generation of Nanobodies as a tool for structural biology

FERNANDO GOLDBAUM; ALZOGARAY V

Córdoba

Congreso; Reunión Anual de SAIB 2016; 2016

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

A promising approach to increase crystal formation and to improve diffraction quality is the use of crystallization chaperones. Nanobodies are exquisite chaperones for crystallizing complex biological systems such as membrane proteins. Nanobodies are the smallest and most stable single-domain fragments with the full antigen-binding capacity and that naturally occur in camelids. Here we describe a general methodology for the generation of nanobodies to be used as crystallization chaperones for the structural investigation of soluble and membrane proteins. We have set up a general pipeline to obtain nanobodies against different immunogens, particularly those of clinical relevance. Some of them are membrane proteins resuspended in detergent buffers and lipid vesicles for preserving their native conformation. The strategy includes the use of phage display technology to select the nanobodies, a step that was carried out with different methods to coat the antigens. We employed solid surface of an ELISA plate and nickel coated plate that allows to spatially orient the target protein. As a result, after two rounds of selection, phages that recognize conformational epitopes were obtained. The nanobodies are expressed in the periplasmic space and sixty specific clones were selected for sequencing by binding assays and their ability to avoid aggregation.