RESUMEN
Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.
Asunto(s)
Camélidos del Nuevo Mundo , Cadenas Pesadas de Inmunoglobulina , Ratones Transgénicos , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Camélidos del Nuevo Mundo/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Lectinas Tipo C/metabolismo , Lectinas Tipo C/inmunología , Lectinas Tipo C/genética , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Inmunoglobulina E/inmunología , Humanos , Dependovirus/genética , Dependovirus/inmunología , Inmunoglobulina G/inmunología , COVID-19/inmunología , Linfocitos B/inmunologíaRESUMEN
The chromatin accessibility complex (CHRAC) is an abundant, evolutionarily conserved nucleosome remodeling machinery able to catalyze histone octamer sliding on DNA. CHRAC differs from the related ACF complex by the presence of two subunits with molecular masses of 14 and 16 kDa, whose structure and function were not known. We determined the structure of Drosophila melanogaster CHRAC14-CHRAC16 by X-ray crystallography at 2.4-angstroms resolution and found that they dimerize via a variant histone fold in a typical handshake structure. In further analogy to histones, CHRAC14-16 contain unstructured N- and C-terminal tail domains that protrude from the handshake structure. A dimer of CHRAC14-16 can associate with the N terminus of ACF1, thereby completing CHRAC. Low-affinity interactions of CHRAC14-16 with DNA significantly improve the efficiency of nucleosome mobilization by limiting amounts of ACF. Deletion of the negatively charged C terminus of CHRAC16 enhances DNA binding 25-fold but leads to inhibition of nucleosome sliding, in striking analogy to the effect of the DNA chaperone HMGB1 on nucleosome sliding. The presence of a surface compatible with DNA interaction and the geometry of an H2A-H2B heterodimer may provide a transient acceptor site for DNA dislocated from the histone surface and therefore facilitate the nucleosome remodeling process.